RNA INTERFERENCE MEDIATED INHIBITION OF MYC AND/OR MYB GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating Myc and/or Myb gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of Myc and/or Myb gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of Myc and/or Myb (e.g., c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb) genes. The small nucleic acid molecules are useful in the treatment of cancer and other diseases and disorders.

This application is a continuation of U.S. patent application Ser. No.10/915,896, filed Aug. 11, 2004, which is a continuation-in-part ofInternational Patent Application No. PCT/US03/05326, filed Feb. 20,2003, which claims the benefit of U.S. Provisional Application No.60/418,655, filed Oct. 15, 2002, and parent U.S. patent application Ser.No. 10/915,896 is also a continuation-in-part of International PatentApplication No. PCT/US04/16390, filed May 24, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/826,966,filed Apr. 16, 2004, which is continuation-in-part of U.S. patentapplication Ser. No. 10/757,803, filed Jan. 14, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/720,448,filed Nov. 24, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/693,059, filed Oct. 23, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/444,853,filed May 23, 2003, which is a continuation-in-part of InternationalPatent Application No. PCT/US03/05346, filed Feb. 20, 2003, and acontinuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing43USCNT”,created on Jul. 17, 2008, which is 192,276 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of Myc and/or Myb geneexpression and/or activity. The present invention is also directed tocompounds, compositions, and methods relating to traits, diseases andconditions that respond to the modulation of expression and/or activityof genes involved in Myc and/or Myb gene expression pathways or othercellular processes that mediate the maintenance or development of suchtraits, diseases and conditions. Specifically, the invention relates tosmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable ofmediating RNA interference (RNAi) against Myc and/or Myb (e.g., c-Myc,N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and/or v-Myb) gene expression. Suchsmall nucleic acid molecules are useful, for example, in providingcompositions for treatment of traits, diseases and conditions that canrespond to modulation of Myc and/or Myb gene expression in a subject,such as a variety of oncogenic and proliferative diseases and disordersincluding various cancers including, but not limited to, multiple drugresistant cancers, such as leukemias including acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), Acute lymphocyticleukemia (ALL), and chronic lymphocytic leukemia; ovarian cancer; breastcancer; cancers of the head and neck; lymphomas such as mantle celllymphoma; non-Hodgkin's lymphoma; Burkitt's lymphoma; adenoma; squamouscell carcinoma; laryngeal carcinoma; multiple myeloma; melanoma;colorectal cancer; prostate cancer; and proliferative diseases such asrestenosis, polycystic kidney disease, and any other indications thatcan respond to the level of Myc and/or Myb in a cell or tissue.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double-strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT. Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certainsingle-stranded siRNA constructs, including certain 5′-phosphorylatedsingle-stranded siRNAs that mediate RNA interference in HeLa cells.Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13,83-105, describe certain chemically and structurally modified siRNAmolecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certainchemically and structurally modified siRNA molecules. Woolf et al.,International PCT Publication Nos. WO 03/064626 and WO 03/064625describe certain chemically-modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating Myc and/or Myb gene expression using short interferingnucleic acid (siNA) molecules. This invention also relates to compounds,compositions, and methods useful for modulating the expression andactivity of other genes involved in pathways of Myc and/or Myb geneexpression and/or activity by RNA interference (RNAi) using smallnucleic acid molecules. In particular, the instant invention featuressmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression of Myc and/or Myb genes (e.g., c-Myc,N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb) and genes involved in theMyc pathway such as cyclin D1 and D2, cyclin E, CDK4, and cdc25A, CDK2and CDK4 genes.

An siNA of the invention can be unmodified or chemically-modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically-modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating Myc and/or Myb geneexpression or activity in cells by RNA interference (RNAi). The use ofchemically-modified siNA improves various properties of native siNAmolecules through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake. Further, contrary to earlierpublished studies, siNA having multiple chemical modifications retainsits RNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, diagnostic,target validation, genomic discovery, genetic engineering, andpharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofMyc genes encoding proteins, such as proteins comprising Myc associatedwith the maintenance and/or development of a variety of oncogenic andproliferative diseases and disorders including various cancersincluding, but not limited to, multiple drug resistant cancers, such asleukemias including acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia; ovarian cancer; breast cancer; cancers ofthe head and neck; lymphomas such as mantle cell lymphoma; non-Hodgkin'slymphoma; Burkitt's lymphoma; adenoma; squamous cell carcinoma;laryngeal carcinoma; multiple myeloma; melanoma; colorectal cancer;prostate cancer; and proliferative diseases such as restenosis,polycystic kidney disease, and any other indications that can respond tothe level of Myc in a cell or tissue, such as genes encoding sequencescomprising those sequences referred to by GenBank Accession Nos. shownin Table I, referred to herein generally as Myc (e.g., c-Myc, N-Myc, andL-Myc).

In another embodiment, the invention features one or more siNA moleculesand methods that independently or in combination modulate the expressionof Myb genes encoding proteins, such as proteins comprising Mybassociated with the maintenance and/or development of a variety ofoncogenic and proliferative diseases and disorders including variouscancers including, but not limited to, multiple drug resistant cancers,such as leukemias including acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia; ovarian cancer; breast cancer; cancers ofthe head and neck; lymphomas such as mantle cell lymphoma; non-Hodgkin'slymphoma; Burkitt's lymphoma; adenoma; squamous cell carcinoma;laryngeal carcinoma; multiple myeloma; melanoma; colorectal cancer;prostate cancer; and proliferative diseases such as restenosis,polycystic kidney disease, and any other indications that can respond tothe level of Myb in a cell or tissue, such as genes encoding sequencescomprising those sequences referred to by GenBank Accession Nos. shownin Table I, referred to herein generally as Myb (e.g., c-Myb, a-Myb,b-Myb, and v-Myb).

The description below of the various aspects and embodiments of theinvention is provided with reference to exemplary Myc and/or Myb genesreferred to herein as Myc and/or Myb. However, the various aspects andembodiments are also directed to other Myc and/or Myb genes, such as Mycand/or Myb homolog genes and transcript variants including c-Myc, N-Myc,L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb, other Myc and Myb genes referredto by Accession number in Table I, and polymorphisms (e.g., singlenucleotide polymorphism, (SNPs)) associated with certain Myc and/or Mybgenes. As such, the various aspects and embodiments are also directed toother genes that are involved in Myc and/or Myb mediated pathways ofsignal transduction or gene expression that are involved, for example,in the progression and/or maintenance of cancer or other proliferativediseases and disorders. These additional genes can be analyzed fortarget sites using the methods described for Myc and/or Myb genesherein. Thus, the modulation of other genes and the effects of suchmodulation of the other genes can be performed, determined, and measuredas described herein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc gene (e.g., c-Myc, N-Myc, and/or L-Myc), wherein said siNAmolecule comprises about 15 to about 28 base pairs.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myb gene (e.g., c-Myb, a-Myb, b-Myb, and/or v-Myb), wherein saidsiNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a MycRNA (e.g., c-Myc, N-Myc, and/or L-Myc RNA) via RNA interference (RNAi),wherein the double-stranded siNA molecule comprises a first and a secondstrand, each strand of the siNA molecule is about 18 to about 28nucleotides in length, the first strand of the siNA molecule comprisesnucleotide sequence having sufficient complementarity to the Myc RNA forthe siNA molecule to direct cleavage of the Myc RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a MybRNA (e.g., c-Myb, a-Myb, b-Myb, and/or v-Myb RNA) via RNA interference(RNAi), wherein the double-stranded siNA molecule comprises a first anda second strand, each strand of the siNA molecule is about 18 to about28 nucleotides in length, the first strand of the siNA moleculecomprises nucleotide sequence having sufficient complementarity to theMyb RNA for the siNA molecule to direct cleavage of the Myb RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a MycRNA (e.g., c-Myc, N-Myc, and/or L-Myc RNA) via RNA interference (RNAi),wherein the double-stranded siNA molecule comprises a first and a secondstrand, each strand of the siNA molecule is about 18 to about 23nucleotides in length, the first strand of the siNA molecule comprisesnucleotide sequence having sufficient complementarity to the Myc RNA forthe siNA molecule to direct cleavage of the Myc RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a MybRNA (e.g., c-Myb, a-Myb, b-Myb, and/or v-Myb RNA) via RNA interference(RNAi), wherein the double-stranded siNA molecule comprises a first anda second strand, each strand of the siNA molecule is about 18 to about23 nucleotides in length, the first strand of the siNA moleculecomprises nucleotide sequence having sufficient complementarity to theMyb RNA for the siNA molecule to direct cleavage of the Myb RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a chemically synthesizeddouble-stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a Myc RNA (e.g., c-Myc, N-Myc, and/or L-Myc RNA) viaRNA interference (RNAi), wherein each strand of the siNA molecule isabout 18 to about 28 nucleotides in length; and one strand of the siNAmolecule comprises nucleotide sequence having sufficient complementarityto the Myc RNA for the siNA molecule to direct cleavage of the Myc RNAvia RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble-stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a Myb RNA (e.g., c-Myb, a-Myb, b-Myb, and/or v-MybRNA) via RNA interference (RNAi), wherein each strand of the siNAmolecule is about 18 to about 28 nucleotides in length; and one strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the Myb RNA for the siNA molecule to direct cleavageof the Myb RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble-stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a Myc RNA (e.g., c-Myc, N-Myc, and/or L-Myc RNA) viaRNA interference (RNAi), wherein each strand of the siNA molecule isabout 18 to about 23 nucleotides in length; and one strand of the siNAmolecule comprises nucleotide sequence having sufficient complementarityto the Myc RNA for the siNA molecule to direct cleavage of the Myc RNAvia RNA interference.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a MybRNA (e.g., c-Myb, a-Myb, b-Myb, and/or v-Myb RNA) via RNA interference(RNAi), wherein the double-stranded siNA molecule comprises a first anda second strand, each strand of the siNA molecule is about 18 to about23 nucleotides in length, the first strand of the siNA moleculecomprises nucleotide sequence having sufficient complementarity to theMyb RNA for the siNA molecule to direct cleavage of the Myb RNA via RNAinterference, and the second strand of said siNA molecule comprisesnucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a Myc gene, for example, wherein the Mycgene comprises Myc encoding sequence.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a Myc gene, for example, wherein the Mycgene comprises Myc non-coding sequence or regulatory elements involvedin Myc gene expression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of Myc genes or a Myc gene family, wherein the genes or genefamily sequences share sequence homology. Such homologous sequences canbe identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering Myc targets that share sequence homology (e.g., c-Myc, N-Myc,L-Myc). As such, one advantage of using siNAs of the invention is that asingle siNA can be designed to include nucleic acid sequence that iscomplementary to the nucleotide sequence that is conserved between thehomologous genes. In this approach, a single siNA can be used to inhibitexpression of more than one gene instead of using more than one siNAmolecule to target the different genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against Myc RNA, wherein the siNA molecule comprises a sequencecomplementary to any RNA having Myc encoding sequence, such as thosesequences having GenBank Accession Nos. shown in Table I. In anotherembodiment, the invention features an siNA molecule having RNAi activityagainst Myc RNA, wherein the siNA molecule comprises a sequencecomplementary to an RNA having variant Myc encoding sequence, forexample other mutant Myc genes not shown in Table I but known in the artto be associated with the maintenance and/or development of cancer orother proliferative diseases and disorders. Chemical modifications asshown in Tables III and IV or otherwise described herein can be appliedto any siNA construct of the invention. In another embodiment, an siNAmolecule of the invention includes a nucleotide sequence that caninteract with nucleotide sequence of a Myc gene and thereby mediatesilencing of Myc gene expression, for example, wherein the siNA mediatesregulation of Myc gene expression by cellular processes that modulatethe chromatin structure or methylation patterns of the Myc gene andprevent transcription of the Myc gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of Myc proteins arising from Mychaplotype polymorphisms that are associated with a disease or condition,(e.g., cancer or other proliferative diseases and disorders). Analysisof Myc genes, or Myc protein or RNA levels can be used to identifysubjects with such polymorphisms or those subjects who are at risk ofdeveloping traits, conditions, or diseases described herein. Thesesubjects are amenable to treatment, for example, treatment with siNAmolecules of the invention and any other composition useful in treatingdiseases related to Myc gene expression. As such, analysis of Mycprotein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of Myc protein orRNA levels can be used to predict treatment outcome and to determine theefficacy of compounds and compositions that modulate the level and/oractivity of certain Myc proteins associated with a trait, condition, ordisease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a Myc protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a Myc gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a Myc protein or a portion thereof. The siNA moleculefurther comprises a sense region, wherein said sense region comprises anucleotide sequence of a Myc gene or a portion thereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a Myc gene. In another embodiment, the invention features ansiNA molecule comprising a region, for example, the antisense region ofthe siNA construct, complementary to a sequence comprising a Myc genesequence or a portion thereof.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a Myb gene, for example, wherein the Mybgene comprises Myb encoding sequence.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a Myb gene, for example, wherein the Mybgene comprises Myb non-coding sequence or regulatory elements involvedin Myb gene expression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of Myb genes or a Myb gene family, wherein the genes or genefamily sequences share sequence homology. Such homologous sequences canbe identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering Myb targets that share sequence homology (e.g., c-Myb, a-Myb,b-Myb, v-Myb). As such, one advantage of using siNAs of the invention isthat a single siNA can be designed to include nucleic acid sequence thatis complementary to the nucleotide sequence that is conserved betweenthe homologous genes. In this approach, a single siNA can be used toinhibit expression of more than one gene instead of using more than onesiNA molecule to target the different genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against Myb RNA, wherein the siNA molecule comprises a sequencecomplementary to any RNA having Myb encoding sequence, such as thosesequences having GenBank Accession Nos. shown in Table I. In anotherembodiment, the invention features an siNA molecule having RNAi activityagainst Myb RNA, wherein the siNA molecule comprises a sequencecomplementary to an RNA having variant Myb encoding sequence, forexample other mutant Myb genes not shown in Table I but known in the artto be associated with the maintenance and/or development of cancer orother proliferative diseases and disorders. Chemical modifications asshown in Tables III and IV or otherwise described herein can be appliedto any siNA construct of the invention. In another embodiment, an siNAmolecule of the invention includes a nucleotide sequence that caninteract with nucleotide sequence of a Myb gene and thereby mediatesilencing of Myb gene expression, for example, wherein the siNA mediatesregulation of Myb gene expression by cellular processes that modulatethe chromatin structure or methylation patterns of the Myb gene andprevent transcription of the Myb gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of Myb proteins arising from Mybhaplotype polymorphisms that are associated with a disease or condition,(e.g., cancer or other proliferative diseases and disorders). Analysisof Myb genes, or Myb protein or RNA levels can be used to identifysubjects with such polymorphisms or those subjects who are at risk ofdeveloping traits, conditions, or diseases described herein. Thesesubjects are amenable to treatment, for example, treatment with siNAmolecules of the invention and any other composition useful in treatingdiseases related to Myb gene expression. As such, analysis of Mybprotein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of Myb protein orRNA levels can be used to predict treatment outcome and to determine theefficacy of compounds and compositions that modulate the level and/oractivity of certain Myb proteins associated with a trait, condition, ordisease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a Myb protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a Myb gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a Myb protein or a portion thereof. The siNA moleculefurther comprises a sense region, wherein said sense region comprises anucleotide sequence of a Myb gene or a portion thereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a Myb gene. In another embodiment, the invention features ansiNA molecule comprising a region, for example, the antisense region ofthe siNA construct, complementary to a sequence comprising a Myb genesequence or a portion thereof.

In one embodiment, the antisense region of Myc siNA constructs comprisesa sequence complementary to sequence having any of SEQ ID NOs. 1-118 or595-598. The antisense region comprises sequence having any of SEQ IDNOs. 119-236, 610-613, 618-621, 626-629, 657, 659, 661, 664, 666, 668,670, or 673. In another embodiment, the sense region of Myc constructscomprises sequence having any of SEQ ID NOs. 1-118, 595-598, 606-609,614-617, 622-625, 656, 658, 660, 662, 663, 665, 667, 669, 671, 672.

In one embodiment, the antisense region of Myb siNA constructs comprisesa sequence complementary to sequence having any of SEQ ID NOs. 237-415,599-602, or 605. The antisense region comprises sequence having any ofSEQ ID NOs. 416-594, 603, 604, 634, 635, 640-643, 648-651, 657, 659,661, 664, 675, 677, 679, or 682. In another embodiment, the sense regionof Myb constructs comprises sequence having any of SEQ ID NOs. 237-415,599-602, 605, 630-633, 636-639, 644-647, 656, 658, 660, 662, 663, 674,676, 678, 680, or 681.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-651 and 656-682. The sequences shown in SEQ ID NOs: 1-651and 656-682 are not limiting. An siNA molecule of the invention cancomprise any contiguous Myc and/or Myb sequence (e.g., about 15 to about25 or more or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or or morecontiguous Myc and/or Myb nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a Myc protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a Myc protein, and wherein said siNA further comprisesa sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, whereinsaid sense region and said antisense region are comprised in a linearmolecule where the sense region comprises at least about 15 nucleotidesthat are complementary to the antisense region.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a Myc gene. Because thec-Myc, N-Myc, and L-Myc genes can share some degree of sequence homologywith each other, siNA molecules can be designed to target a class of Mycgenes or alternately specific Myc genes (e.g., polymorphic variants) byselecting sequences that are either shared amongst different Myc targetsor alternatively that are unique for a specific Myc target. Therefore,in one embodiment, the siNA molecule can be designed to target conservedregions of Myc RNA sequences having homology among several Myc genevariants so as to target a class of Myc genes with one siNA molecule.Accordingly, in one embodiment, the siNA molecule of the inventionmodulates the expression of one or both Myc alleles in a subject. Inanother embodiment, the siNA molecule can be designed to target asequence that is unique to a specific Myc RNA sequence (e.g., a singleMyc allele or Myc single nucleotide polymorphism (SNP)) due to the highdegree of specificity that the siNA molecule requires to mediate RNAiactivity.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a Myb protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a Myb protein, and wherein said siNA further comprisesa sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, whereinsaid sense region and said antisense region are comprised in a linearmolecule where the sense region comprises at least about 15 nucleotidesthat are complementary to the antisense region.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a Myb gene. Because thec-Myb, a-Myb, b-Myb, and v-Myb genes can share some degree of sequencehomology with each other, siNA molecules can be designed to target aclass of Myb genes or alternately specific Myb genes (e.g., polymorphicvariants) by selecting sequences that are either shared amongstdifferent Myb targets or alternatively that are unique for a specificMyb target. Therefore, in one embodiment, the siNA molecule can bedesigned to target conserved regions of Myb RNA sequences havinghomology among several Myb gene variants so as to target a class of Mybgenes with one siNA molecule. Accordingly, in one embodiment, the siNAmolecule of the invention modulates the expression of one or both Myballeles in a subject. In another embodiment, the siNA molecule can bedesigned to target a sequence that is unique to a specific Myb RNAsequence (e.g., a single Myb allele or Myb single nucleotidepolymorphism (SNP)) due to the high degree of specificity that the siNAmolecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for Myc and/orMyb expressing nucleic acid molecules, such as RNA encoding a Myc and/orMyb protein. In one embodiment, the invention features a RNA based siNAmolecule (e.g., an siNA molecule comprising 2′-OH nucleotides) havingspecificity for Myc and/or Myb expressing nucleic acid molecules thatincludes one or more chemical modifications described herein.Non-limiting examples of such chemical modifications include withoutlimitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications (e.g., RNAbased siNA constructs), when used in various siNA constructs, are shownto preserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds. Furthermore, contraryto the data published by Parrish et al., supra, applicant demonstratesthat multiple (greater than one) phosphorothioate substitutions arewell-tolerated and confer substantial increases in serum stability formodified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single-stranded, the percent modification can be based uponthe total number of nucleotides present in the single-stranded siNAmolecules. Likewise, if the siNA molecule is double-stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a Mycand/or Myb gene. In one embodiment, the double-stranded siNA moleculecomprises one or more chemical modifications and each strand of thedouble-stranded siNA is about 21 nucleotides long. In one embodiment,the double-stranded siNA molecule does not contain any ribonucleotides.In another embodiment, the double-stranded siNA molecule comprises oneor more ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule independently comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides, wherein each strand comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides that are complementary to the nucleotides of theother strand. In one embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence or a portion thereof of the Mycand/or Myb gene, and the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence of the Myc and/or Myb gene or a portion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene comprising an antisense region, wherein theantisense region comprises a nucleotide sequence that is complementaryto a nucleotide sequence of the Myc and/or Myb gene or a portionthereof, and a sense region, wherein the sense region comprises anucleotide sequence substantially similar to the nucleotide sequence ofthe Myc and/or Myb gene or a portion thereof. In one embodiment, theantisense region and the sense region independently comprise about 15 toabout 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) nucleotides, wherein the antisense region comprisesabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary tonucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by the Mycand/or Myb gene or a portion thereof and the sense region comprises anucleotide sequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 28” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of adouble-stranded siNA molecule having no overhanging nucleotides. The twostrands of a double-stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. The sense region can be connected to the antisense regionvia a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc gene, wherein the siNA molecule comprises about 15 to about 30(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30) base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aMyc gene or a portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe Myc gene. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a Myc gene or portion thereof,and the second strand of the double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence orportion thereof of the Myc gene. In another embodiment, each strand ofthe siNA molecule comprises about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,and each strand comprises at least about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to the nucleotides of the otherstrand. The Myc gene can comprise, for example, sequences referred to inTable I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myb gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. The sense region can be connected to the antisense regionvia a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myb gene, wherein the siNA molecule comprises about 15 to about 30(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30) base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aMyb gene or a portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe Myb gene. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a Myb gene or portion thereof,and the second strand of the double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence orportion thereof of the Myb gene. In another embodiment, each strand ofthe siNA molecule comprises about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,and each strand comprises at least about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to the nucleotides of the otherstrand. The Myb gene can comprise, for example, sequences referred to inTable I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a Myc gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the Myc gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The Myc gene can comprise, for example, sequences referred to in TableI. In another embodiment, the siNA is a double-stranded nucleic acidmolecule, where each of the two strands of the siNA moleculeindependently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36,37, 38, 39, or 40) nucleotides, and where one of the strands of the siNAmolecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20,21, 22, 23, 24 or 25 or more) nucleotides that are complementary to thenucleic acid sequence of the Myc gene or a portion thereof.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a Myc gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The Myc genecan comprise, for example, sequences referred in to Table I.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a Myb gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the Myb gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The Myb gene can comprise, for example, sequences referred to in TableI. In another embodiment, the siNA is a double-stranded nucleic acidmolecule, where each of the two strands of the siNA moleculeindependently comprise about 0.15 to about 40 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35,36, 37, 38, 39, or 40) nucleotides, and where one of the strands of thesiNA molecule comprises at least about 15 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are complementaryto the nucleic acid sequence of the Myb gene or a portion thereof.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a Myb gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The Myb genecan comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by the Mycand/or Myb gene or a portion thereof and the sense region comprises anucleotide sequence that is complementary to the antisense region, andwherein the siNA molecule has one or more modified pyrimidine and/orpurine nucleotides. In one embodiment, the pyrimidine nucleotides in thesense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In one embodiment, thepyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in theantisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. Inanother embodiment of any of the above-described siNA molecules, anynucleotides present in a non-complementary region of the sense strand(e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene, wherein the siNA molecule is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule, and wherein the fragment comprising the senseregion includes a terminal cap moiety at the 5′-end, the 3′-end, or bothof the 5′ and 3′ ends of the fragment. In one embodiment, the terminalcap moiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In anotherembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39,or 40) nucleotides. In a non-limiting example, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of lengthbetween about 15 to about 40 nucleotides in length. In one embodiment,all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides. In one embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In oneembodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-deoxy-2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene comprising a sense region and an antisenseregion, wherein the antisense region comprises a nucleotide sequencethat is complementary to a nucleotide sequence of RNA encoded by the Mycand/or Myb gene or a portion thereof and the sense region comprises anucleotide sequence that is complementary to the antisense region, andwherein the purine nucleotides present in the antisense region comprise2′-deoxy-purine nucleotides. In an alternative embodiment, the purinenucleotides present in the antisense region comprise 2′-O-methyl purinenucleotides. In either of the above embodiments, the antisense regioncan comprise a phosphorothioate internucleotide linkage at the 3′ end ofthe antisense region. Alternatively, in either of the above embodiments,the antisense region can comprise a glyceryl modification at the 3′ endof the antisense region. In another embodiment of any of theabove-described siNA molecules, any nucleotides present in anon-complementary region of the antisense strand (e.g. overhang region)are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a Myc and/orMyb transcript having sequence unique to a particular Myc and/or Mybdisease related allele, such as sequence comprising a single nucleotidepolymorphism (SNP) associated with the disease specific allele. As such,the antisense region of an siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a Myc and/or Myb gene, wherein the siNA molecule is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. In another embodiment, the siNA molecule is adouble-stranded nucleic acid molecule, where each strand is about 21nucleotides long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region, where about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the Myc and/or Myb gene. In another embodiment, about 21nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the Myc and/or Mybgene. In any of the above embodiments, the 5′-end of the fragmentcomprising said antisense region can optionally includes a phosphategroup.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa Myc and/or Myb RNA sequence (e.g., wherein said target RNA sequence isencoded by a Myc and/or Myb gene involved in the Myc and/or Mybpathway), wherein the siNA molecule does not contain any ribonucleotidesand wherein each strand of the double-stranded siNA molecule is about 15to about 30 nucleotides. In one embodiment, the siNA molecule is 21nucleotides in length. Examples of non-ribonucleotide containing siNAconstructs are combinations of stabilization chemistries shown in TableIV in any combination of Sense/Antisense chemistries, such as Stab 7/8,Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab18/20.

In one embodiment, the invention features a chemically synthesizeddouble-stranded RNA molecule that directs cleavage of a Myc and/or MybRNA via RNA interference, wherein each strand of said RNA molecule isabout 15 to about 30 nucleotides in length; one strand of the RNAmolecule comprises nucleotide sequence having sufficient complementarityto the Myc and/or Myb RNA for the RNA molecule to direct cleavage of theMyc and/or Myb RNA via RNA interference; and wherein at least one strandof the RNA molecule optionally comprises one or more chemically-modifiednucleotides described herein, such as without limitationdeoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a Myc and/or Myb gene, whereinthe siNA molecule comprises one or more chemical modifications and eachstrand of the double-stranded siNA is independently about 15 to about 30or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 or more) nucleotides long. In one embodiment, the siNAmolecule of the invention is a double-stranded nucleic acid moleculecomprising one or more chemical modifications, where each of the twofragments of the siNA molecule independently comprise about 15 to about40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and whereone of the strands comprises at least 15 nucleotides that arecomplementary to nucleotide sequence of Myc and/or Myb encoding RNA or aportion thereof. In a non-limiting example, each of the two fragments ofthe siNA molecule comprise about 21 nucleotides. In another embodiment,the siNA molecule is a double-stranded nucleic acid molecule comprisingone or more chemical modifications, where each strand is about 21nucleotide long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acid moleculecomprising one or more chemical modifications, where each strand isabout 19 nucleotide long and where the nucleotides of each fragment ofthe siNA molecule are base-paired to the complementary nucleotides ofthe other fragment of the siNA molecule to form at least about 15 (e.g.,15, 16, 17, 18, or 19) base pairs, wherein one or both ends of the siNAmolecule are blunt ends. In one embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule is a2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In anotherembodiment, all nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule. In another embodiment, the siNA molecule is adouble-stranded nucleic acid molecule of about 19 to about 25 base pairshaving a sense region and an antisense region and comprising one or morechemical modifications, where about 19 nucleotides of the antisenseregion are base-paired to the nucleotide sequence or a portion thereofof the RNA encoded by the Myc and/or Myb gene. In another embodiment,about 21 nucleotides of the antisense region are base-paired to thenucleotide sequence or a portion thereof of the RNA encoded by the Mycand/or Myb gene. In any of the above embodiments, the 5′-end of thefragment comprising said antisense region can optionally include aphosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits, downregulates, or reduces expression of a Myc and/or Myb gene, wherein oneof the strands of the double-stranded siNA molecule is an antisensestrand which comprises nucleotide sequence that is complementary tonucleotide sequence of Myc and/or Myb RNA or a portion thereof, theother strand is a sense strand which comprises nucleotide sequence thatis complementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down regulates,or reduces expression of a Myc and/or Myb gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of Myc and/or Myb RNA or a portion thereof, wherein the otherstrand is a sense strand which comprises nucleotide sequence that iscomplementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down regulates,or reduces expression of a Myc and/or Myb gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of Myc and/or Myb RNA that encodes a protein or portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification. In oneembodiment, each strand of the siNA molecule comprises about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises atleast about 15 nucleotides that are complementary to the nucleotides ofthe other strand. In one embodiment, the siNA molecule is assembled fromtwo oligonucleotide fragments, wherein one fragment comprises thenucleotide sequence of the antisense strand of the siNA molecule and asecond fragment comprises nucleotide sequence of the sense region of thesiNA molecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aMyc and/or Myb gene, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, each of the two strands of the siNA molecule can compriseabout 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In oneembodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotidesof each strand of the siNA molecule are base-paired to the complementarynucleotides of the other strand of the siNA molecule. In anotherembodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotidesof each strand of the siNA molecule are base-paired to the complementarynucleotides of the other strand of the siNA molecule, wherein at leasttwo 3′ terminal nucleotides of each strand of the siNA molecule are notbase-paired to the nucleotides of the other strand of the siNA molecule.In another embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as2′-deoxy-thymidine. In one embodiment, each strand of the siNA moleculeis base-paired to the complementary nucleotides of the other strand ofthe siNA molecule. In one embodiment, about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides of the antisense strand are base-paired to the nucleotidesequence of the Myc and/or Myb RNA or a portion thereof. In oneembodiment, about 18 to about 25 (e.g., about 18, 19, 20, 21, 22, 23,24, or 25) nucleotides of the antisense strand are base-paired to thenucleotide sequence of the Myc and/or Myb RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aMyc and/or Myb gene, wherein one of the strands of the double-strandedsiNA molecule is an antisense strand which comprises nucleotide sequencethat is complementary to nucleotide sequence of Myc and/or Myb RNA or aportion thereof, the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification, and wherein the 5′-end of the antisense strandoptionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aMyc and/or Myb gene, wherein one of the strands of the double-strandedsiNA molecule is an antisense strand which comprises nucleotide sequencethat is complementary to nucleotide sequence of Myc and/or Myb RNA or aportion thereof, the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification, and wherein the nucleotide sequence or a portionthereof of the antisense strand is complementary to a nucleotidesequence of the untranslated region or a portion thereof of the Mycand/or Myb RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aMyc and/or Myb gene, wherein one of the strands of the double-strandedsiNA molecule is an antisense strand which comprises nucleotide sequencethat is complementary to nucleotide sequence of Myc and/or Myb RNA or aportion thereof, wherein the other strand is a sense strand whichcomprises nucleotide sequence that is complementary to a nucleotidesequence of the antisense strand, wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the Myc and/or MybRNA or a portion thereof that is present in the Myc and/or Myb RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding Myc and/or Myband the sense region can comprise sequence complementary to theantisense region. The siNA molecule can comprise two distinct strandshaving complementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides comprising a backbone modified internucleotide linkagehaving Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides or non-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises a 5′-terminal phosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises one or more phosphorothioate internucleotide linkages. Forexample, in a non-limiting example, the invention features achemically-modified short interfering nucleic acid (siNA) having about1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkagesin one siNA strand. In yet another embodiment, the invention features achemically-modified short interfering nucleic acid (siNA) individuallyhaving about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in both siNA strands. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the siNA duplex, for example in the sense strand, theantisense strand, or both strands. The siNA molecules of the inventioncan comprise one or more phosphorothioate internucleotide linkages atthe 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sensestrand, the antisense strand, or both strands. For example, an exemplarysiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, an siNA molecule of the invention comprises asingle-stranded hairpin structure, wherein the siNA is about 36 to about70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification comprising a structure having any ofFormulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., about 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically-modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically-modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double-stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically-modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3-OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of an siNA molecule of the invention. For example,chemically-modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) can be present at the 3′-end, the 5′-end, or bothof the 3′ and 5′-ends of the antisense strand, the sense strand, or bothantisense and sense strands of the siNA molecule. In one embodiment, thechemically-modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the 5′-end and 3′-end of the sensestrand and the 3′-end of the antisense strand of a double-stranded siNAmolecule of the invention. In one embodiment, the chemically-modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the terminal position of the 5′-end and 3′-end of thesense strand and the 3′-end of the antisense strand of a double-strandedsiNA molecule of the invention. In one embodiment, thechemically-modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the two terminal positions of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double-stranded siNA molecule of the invention. In oneembodiment, the chemically-modified nucleoside or non-nucleoside (e.g.,a moiety having Formula V, VI or VII) is present at the penultimateposition of the 5′-end and 3′-end of the sense strand and the 3′-end ofthe antisense strand of a double-stranded siNA molecule of theinvention. In addition, a moiety having Formula VII can be present atthe 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against Myc and/or Myb inside a cellor reconstituted in vitro system comprising a sense region, wherein oneor more pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of thesechemically-modified siNAs are shown in FIGS. 4, 5, and 22 and Tables IIIand IV herein. In any of these described embodiments, the purinenucleotides present in the sense region are alternatively 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides) and one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality of purine nucleotides arepurine ribonucleotides) and any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such,chemically-modified nucleotides present in the siNA molecules of theinvention, preferably in the antisense strand of the siNA molecules ofthe invention, but also optionally in the sense and/or both antisenseand sense strands, are resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi. Non-limitingexamples of nucleotides having a Northern configuration include lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides.

In one embodiment, the sense strand of a double-stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against Myc and/or Myb inside a cell orreconstituted in vitro system, wherein the chemical modificationcomprises a conjugate covalently attached to the chemically-modifiedsiNA molecule. Non-limiting examples of conjugates contemplated by theinvention include conjugates and ligands described in Vargeese et al.,U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by referenceherein in its entirety, including the drawings. In another embodiment,the conjugate is covalently attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically-modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single-stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single-stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single-stranded siNAmolecule of the invention comprises about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, the single-stranded siNAmolecule of the invention comprises one or more chemically-modifiednucleotides or non-nucleotides described herein. For example, all thepositions within the siNA molecule can include chemically-modifiednucleotides such as nucleotides having any of Formulae I-VII, or anycombination thereof to the extent that the ability of the siNA moleculeto support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end and/or the5′-end. The siNA optionally further comprises about 1 to about 4 or more(e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the3′-end of the siNA molecule, wherein the terminal nucleotides canfurther comprise one or more (e.g., 1, 2, 3, 4 or more)phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages, and wherein the siNA optionally furthercomprises a terminal phosphate group, such as a 5′-terminal phosphategroup. In any of these embodiments, any purine nucleotides present inthe antisense region are alternatively 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA (i.e., purine nucleotides present in the senseand/or antisense region) can alternatively be locked nucleic acid (LNA)nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides oralternately a plurality of purine nucleotides are LNA nucleotides).Also, in any of these embodiments, any purine nucleotides present in thesiNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-methoxyethyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single-stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically-modified nucleotides present in the single-strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, an siNA molecule of the invention compriseschemically-modified nucleotides or non-nucleotides (e.g., having any ofFormulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides) at alternating positions within one or more strands orregions of the siNA molecule. For example, such chemical modificationscan be introduced at every other position of a RNA based siNA molecule,starting at either the first or second nucleotide from the 3′-end or5′-end of the siNA. In a non-limiting example, a double-stranded siNAmolecule of the invention in which each strand of the siNA is 21nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 and 21 of each strand are chemically-modified (e.g., withcompounds having any of Formulae I-VII, such as such as 2′-deoxy,2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). In another non-limitingexample, a double-stranded siNA molecule of the invention in which eachstrand of the siNA is 21 nucleotides in length is featured whereinpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand arechemically-modified (e.g., with compounds having any of Formulae I-VII,such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides). Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating theexpression of a Myc and/or Myb gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the Myc and/or Myb gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a Myc and/or Myb gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene and wherein thesense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the Myc and/or Myb gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one Myc and/or Myb gene within a cellcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb genes; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate the expression of the Myc and/or Myb genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more Myc and/or Myb genes within a cellcomprising: (a) synthesizing one or more siNA molecules of theinvention, which can be chemically-modified, wherein the siNA strandscomprise sequences complementary to RNA of the Myc and/or Myb genes andwherein the sense strand sequences of the siNAs comprise sequencesidentical or substantially similar to the sequences of the target RNAs;and (b) introducing the siNA molecules into a cell under conditionssuitable to modulate the expression of the Myc and/or Myb genes in thecell.

In another embodiment, the invention features a method for modulatingthe expression of more than one Myc and/or Myb gene within a cellcomprising: (a) synthesizing an siNA molecule of the invention, whichcan be chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene and wherein thesense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the Myc and/or Myb genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the Myc and/or Myb gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of the Mycand/or Myb gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene and wherein thesense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the Myc and/or Myb gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of the Mycand/or Myb gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one Myc and/or Myb gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb genes; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the Myc and/or Myb genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of the Mycand/or Myb genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a subject or an organismcomprising: (a) synthesizing an siNA molecule of the invention, whichcan be chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the Myc and/or Myb gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the Myc and/or Mybgene in the subject or organism. The level of Myc and/or Myb protein orRNA can be determined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one Myc and/or Myb gene in a subject or anorganism comprising: (a) synthesizing siNA molecules of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the Myc and/or Myb genes;and (b) introducing the siNA molecules into the subject or organismunder conditions suitable to modulate the expression of the Myc and/orMyb genes in the subject or organism. The level of Myc and/or Mybprotein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a Myc and/or Myb gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the Myc and/or Myb gene; and(b) introducing the siNA molecule into a cell under conditions suitableto modulate the expression of the Myc and/or Myb gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one Myc and/or Myb gene within a cellcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the Myc and/or Myb gene; and(b) contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate the expression of the Myc and/or Mybgenes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the Myc and/or Myb gene; and(b) contacting a cell of the tissue explant derived from a particularsubject or organism with the siNA molecule under conditions suitable tomodulate the expression of the Myc and/or Myb gene in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the subject or organism the tissue wasderived from or into another subject or organism under conditionssuitable to modulate the expression of the Myc and/or Myb gene in thatsubject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one Myc and/or Myb gene in a tissue explantcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the Myc and/or Myb gene; and(b) introducing the siNA molecules into a cell of the tissue explantderived from a particular subject or organism under conditions suitableto modulate the expression of the Myc and/or Myb genes in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the subject or organism the tissue wasderived from or into another subject or organism under conditionssuitable to modulate the expression of the Myc and/or Myb genes in thatsubject or organism.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a subject or an organismcomprising: (a) synthesizing an siNA molecule of the invention, whichcan be chemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the Myc and/or Myb gene; and(b) introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the Myc and/or Mybgene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one Myc and/or Myb gene in a subject or anorganism comprising: (a) synthesizing siNA molecules of the invention,which can be chemically-modified, wherein the siNA comprises asingle-stranded sequence having complementarity to RNA of the Myc and/orMyb gene; and (b) introducing the siNA molecules into the subject ororganism under conditions suitable to modulate the expression of the Mycand/or Myb genes in the subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a Myc and/or Myb gene in a subject or an organismcomprising contacting the subject or organism with an siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe Myc and/or Myb gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing one or more of a variety of oncogenic and proliferativediseases and disorders including various cancers including but notlimited to multiple drug resistant cancers, such as leukemias includingacute myelogenous leukemia (AML), chronic myelogenous leukemia (CML),Acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia;ovarian cancer, breast cancer, cancers of the head and neck, lymphomassuch as mantle cell lymphoma, non-Hodgkin's lymphoma, Burkitt'slymphoma, adenoma, squamous cell carcinoma, laryngeal carcinoma,multiple myeloma, melanoma, colorectal cancer, prostate cancer; andproliferative diseases such as restenosis, polycystic kidney disease andany other indications that can respond to the level of Myc and/or Myb ina cell or tissue in a subject or an organism comprising contacting thesubject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the Myc and/or Mybgene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one Myc and/or Myb genes in a subject or anorganism comprising contacting the subject or organism with one or moresiNA molecules of the invention under conditions suitable to modulatethe expression of the Myc and/or Myb genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., Myc and/or Myb) gene expression through RNAitargeting of a variety of RNA molecules. In one embodiment, the siNAmolecules of the invention are used to target various RNAs correspondingto a target gene. Non-limiting examples of such RNAs include messengerRNA (mRNA), alternate RNA splice variants of target gene(s),post-transcriptionally modified RNA of target gene(s), pre-mRNA oftarget gene(s), and/or RNA templates. If alternate splicing produces afamily of transcripts that are distinguished by usage of appropriateexons, the instant invention can be used to inhibit gene expressionthrough the appropriate exons to specifically inhibit or to distinguishamong the functions of gene family members. For example, a protein thatcontains an alternatively spliced transmembrane domain can be expressedin both membrane bound and secreted forms. Use of the invention totarget the exon containing the transmembrane domain can be used todetermine the functional consequences of pharmaceutical targeting ofmembrane bound as opposed to the secreted form of the protein.Non-limiting examples of applications of the invention relating totargeting these RNA molecules include therapeutic pharmaceuticalapplications, pharmaceutical discovery applications, moleculardiagnostic and gene function applications, and gene mapping, for exampleusing single nucleotide polymorphism mapping with siNA molecules of theinvention. Such applications can be implemented using known genesequences or from partial sequences available from an expressed sequencetag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as Myc and/or Myb family genes. As such, siNA moleculestargeting multiple Myc and/or Myb targets can provide increasedtherapeutic effect. In addition, siNA can be used to characterizepathways of gene function in a variety of applications. For example, thepresent invention can be used to inhibit the activity of target gene(s)in a pathway to determine the function of uncharacterized gene(s) ingene function analysis, mRNA function analysis, or translationalanalysis. The invention can be used to determine potential target genepathways involved in various diseases and conditions towardpharmaceutical development. The invention can be used to understandpathways of gene expression involved in, for example, the progressionand/or maintenance of cancer or other proliferative diseases anddisorders.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession Nos., for example, Myc and/or Myb genes encodingRNA sequence(s) referred to herein by Genbank Accession number, forexample, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, Northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4N, where N represents the numberof base paired nucleotides in each of the siNA construct strands (e.g.,for an siNA construct having 21 nucleotide sense and antisense strandswith 19 base pairs, the complexity would be 4¹⁹); and (b) assaying thesiNA constructs of (a) above, under conditions suitable to determineRNAi target sites within the target Myc and/or Myb RNA sequence. Inanother embodiment, the siNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described in Example 7 herein. In anotherembodiment, the assay can comprise a cell culture system in which targetRNA is expressed. In another embodiment, fragments of Myc and/or Myb RNAare analyzed for detectable levels of cleavage, for example, by gelelectrophoresis, Northern blot analysis, or RNAse protection assays, todetermine the most suitable target site(s) within the target Myc and/orMyb RNA sequence. The target Myc and/or Myb RNA sequence can be obtainedas is known in the art, for example, by cloning and/or transcription forin vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of target RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,Northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target RNA sequence. The targetRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for preventingor treating one or more of a variety of oncogenic and proliferativediseases and disorders in a subject comprising administering to thesubject a composition of the invention under conditions suitable for theprevention or treatment of one or more of a variety of oncogenic andproliferative diseases and disorders in the subject. In yet anotherembodiment, the invention features a method for reducing or preventingtissue rejection in a subject comprising administering to the subject acomposition of the invention under conditions suitable for the reductionor prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating aMyc and/or Myb gene target, comprising: (a) synthesizing an siNAmolecule of the invention, which can be chemically-modified, wherein oneof the siNA strands includes a sequence complementary to RNA of a Mycand/or Myb target gene; (b) introducing the siNA molecule into a cell, atissue, a subject, or an organism under conditions suitable formodulating expression of the Myc and/or Myb target gene in the cell,tissue, subject, or organism; and (c) determining the function of thegene by assaying for any phenotypic change in the cell, tissue, subject,or organism.

In another embodiment, the invention features a method for validating aMyc and/or Myb target comprising: (a) synthesizing an siNA molecule ofthe invention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a Myc and/or Mybtarget gene; (b) introducing the siNA molecule into a biological systemunder conditions suitable for modulating expression of the Myc and/orMyb target gene in the biological system; and (c) determining thefunction of the gene by assaying for any phenotypic change in thebiological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell, atissue, a subject, or an organism, or extract thereof. The termbiological system also includes reconstituted RNAi systems that can beused in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a Myc and/or Myb target gene in abiological system, including, for example, in a cell, a tissue, asubject, or an organism. In another embodiment, the invention features akit containing more than one siNA molecule of the invention, which canbe chemically-modified, that can be used to modulate the expression ofmore than one Myc and/or Myb target gene in a biological system,including, for example, in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications, for example, one or more chemicalmodifications having any of Formulae I-VII or any combination thereofthat increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formulae I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., have attenuatedor no immunostimulatory properties) comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table IV) or any combination thereof into an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table IV) or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

By “improved toxicologic profile”, is meant that the chemically-modifiedsiNA construct exhibits decreased toxicity in a cell, subject, ororganism compared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In a non-limiting example, siNA molecules withimproved toxicologic profiles are associated with a decreased orattenuated immunostimulatory response in a cell, subject, or organismcompared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In one embodiment, an siNA molecule with animproved toxicological profile comprises no ribonucleotides. In oneembodiment, an siNA molecule with an improved toxicological profilecomprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4ribonucleotides). In one embodiment, an siNA molecule with an improvedtoxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab25, Stab 26, Stab 27, Stab 28, or any combination thereof (see TableIV). In one embodiment, the level of immunostimulatory responseassociated with a given siNA molecule can be measured as is known in theart, for example by determining the level of PKR/interferon response,proliferation, B-cell activation, and/or cytokine production in assaysto quantitate the immunostimulatory response of particular siNAmolecules (see, for example, Leifer et al., 2003, J. Immunother. 26,313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety byreference).

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formulae I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that modulate thepolymerase activity of a cellular polymerase capable of generatingadditional endogenous siNA molecules having sequence homology to thechemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaeI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against Myc and/or Myb in a cell, whereinthe chemical modifications do not significantly effect the interactionof siNA with a target RNA molecule, DNA molecule and/or proteins orother factors that are essential for RNAi in a manner that woulddecrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against Myc and/or Mybcomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against Myc and/orMyb target RNA comprising (a) introducing nucleotides having any ofFormulae I-VII or any combination thereof into an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved RNAi activity against thetarget RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against Myc and/orMyb target DNA comprising (a) introducing nucleotides having any ofFormulae I-VII or any combination thereof into an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved RNAi activity against thetarget DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against Myc and/or Myb with improved cellular uptakecomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against Myc and/or Myb, wherein the siNA construct comprises one ormore chemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically-modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” chemistries and variantsthereof (see Table IV) wherein the 5′-end and 3′-end of the sense strandof the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically-modified siNA molecules of step (c) underconditions suitable for isolating chemically-modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically-modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically-modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically-modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4, 5, 6, and 22, andTables II and III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex ordouble-stranded structure, for example wherein the double-strandedregion is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strandcomprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense strand comprises nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof (e.g., about 15 to about 25or more nucleotides of the siNA molecule are complementary to the targetnucleic acid or a portion thereof). Alternatively, the siNA is assembledfrom a single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single-stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single-stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al.,U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCTApplication No. US04/16390, filed May 24, 2004). The multifunctionalsiNA of the invention can comprise sequence targeting, for example, tworegions of Myc and/or Myb RNA (see for example target sequences inTables II and III).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically-modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of asubject, an organism, or a cell, by intervening in cellular processessuch as genetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AANI-amino symmetric, AA N7-amino symmetric, GA N7-NI amino-carbonyl,GA+carbonyl-amino N7-NI, GG N1-carbonyl symmetric, GG N3-aminosymmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3,AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AUamino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CCcarbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GGamino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GUcarbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl,UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3,GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “Myc” as used herein is meant Myc protein, peptide, or polypeptidehaving Myc activity, such as encoded by Myc Genbank Accession Nos. shownin Table I or any other Myc transcript derived from a Myc gene. The termMyc also refers to nucleic acid sequences encoding any Myc protein,peptide, or polypeptide having Myc activity. The term “Myc” is alsomeant to include other Myc encoding sequence, such as Myc isoforms(e.g., c-Myc, N-Myc, L-Myc, etc.), mutant Myc genes, splice variants ofMyc genes, and Myc gene polymorphisms.

By “Myb” as used herein is meant Myb protein, peptide, or polypeptidehaving Myb activity, such as encoded by Myb Genbank Accession Nos. shownin Table I or any other Myb transcript derived from a Myb gene. The termMyb also refers to nucleic acid sequences encoding any Myb protein,peptide, or polypeptide having Myb activity. The term “Myb” is alsomeant to include other Myb encoding sequence, such as Myb isoforms(e.g., c-Myb, a-Myb, b-Myb, v-Myb, etc.), mutant Myb genes, splicevariants of Myb genes, and Myb gene polymorphisms.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80%, etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, an siNA moleculeof the invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof).

In one embodiment, siNA molecules of the invention that down regulate orreduce Myc and/or Myb gene expression are used for preventing ortreating, in a subject or organism, a variety of oncogenic andproliferative diseases and disorders. By “proliferative disease” or“cancer” as used herein is meant, any disease or condition characterizedby unregulated cell growth or replication as is known in the art,including various cancers including but not limited to multiple drugresistant cancers, such as leukemias including acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), Acute lymphocyticleukemia (ALL), and chronic lymphocytic leukemia; ovarian cancer, breastcancer, cancers of the head and neck, lymphomas such as mantle celllymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, adenoma, squamouscell carcinoma, laryngeal carcinoma, multiple myeloma, melanoma,colorectal cancer, prostate cancer; and proliferative diseases such asrestenosis, polycystic kidney disease and any other indications that canrespond to the level of Myc and/or Myb in a cell or tissue. In anotherembodiment, the siNA molecules of the invention are used for reducing orpreventing tissue rejection in a subject or organism.

In one embodiment, the siNA molecules of the invention are used to treataffecting the progression and/or maintenance of cancer or otherproliferative diseases and disorders in a subject or organism.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4, 5, and 22.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4, 5, and 22.Examples of such nucleic acid molecules consist essentially of sequencesdefined in these tables and figures. Furthermore, thechemically-modified constructs described in Table IV can be applied toany siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides; and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating diseases or conditions discussed herein (e.g.,cancer) in a subject or an organism. For example, the siNA molecules canbe administered to a subject or can be administered to other appropriatecells evident to those skilled in the art, individually or incombination with one or more drugs under conditions suitable for thetreatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat diseases or conditionsdiscussed herein (e.g., cancer) in a subject or an organism. Forexample, the described molecules could be used in combination with oneor more known compounds, treatments, or procedures to prevent or treatdiseases or conditions (e.g., cancer) in a subject or an organism as areknown in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4 A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a c-Myc siNA sequence. Such chemicalmodifications can be applied to any Myc sequence and/or Myc polymorphismsequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined Myc and/or Myb target sequence, wherein thesense region comprises, for example, about 19, 20, 21, or 22 nucleotides(N) in length, which is followed by a loop sequence of defined sequence(X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a Myc and/orMyb target sequence and having self-complementary sense and antisenseregions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined Myc and/or Myb target sequence, wherein thesense region comprises, for example, about 19, 20, 21, or 22 nucleotides(N) in length, and which is followed by a 3′-restriction site (R2) whichis adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically-modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically-modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double-stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allows the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double-strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically-modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically-modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 22A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a c-Myb siNA sequence.

FIG. 23 shows a non-limiting example of reduction of Myc (c-Myc) mRNA in293T cells mediated by chemically-modified siNAs that target c-Myc mRNA.293T cells were transfected with 0.25 ug/well of lipid complexed with 25nM siNA. A screen of siNA constructs comprising ribonucleotides and3′-terminal dithymidine caps was compared to untreated cells, scrambledsiNA control constructs (Scram1 and Scram2), and cells transfected withlipid alone (transfection control). As shown in the figure, three of thesiNA constructs (Compound number 30993/31069; Compound number30995/31071; and Compound number 30996/31072) show significant reductionof c-Myc RNA expression.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; I-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to prevent ortreat cancer, restenosis, polycystic kidney disease, or any other trait,disease or condition that is related to or will respond to the levels ofMyc and/or Myb in a cell or tissue, alone or in combination with othertherapies. For example, an siNA molecule can comprise a deliveryvehicle, including liposomes, for administration to a subject, carriersand diluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations. Methods for the delivery of nucleic acidmolecules are described in Akhtar et al., 1992, Trends Cell Bio., 2,139; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed.Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192, all of which areincorporated herein by reference. Beigelman et al., U.S. Pat. No.6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). Alternatively, thenucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

In another embodiment, the nucleic acid molecules of the invention canalso be formulated or complexed with polyethyleneimine and derivativesthereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient:

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7;Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al.,1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol.,10, 4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

Myc Biology and Biochemistry

Significant progress has been made over the last two decades in ourunderstanding of the function of c-Myc in normal cells and in cancercells. In contrast to the tightly regulated c-Myc gene in normal cells,which only express the gene when cells actively divide, c-Myc in cancercells may be expressed in an uncontrolled fashion as the result ofgenetic aberrations. It is now well established that the deregulatedexpression of c-Myc plays a significant role in human cancerdevelopment. The c-Myc protein or the c-Myc gene is overexpressed in awide variety of human cancers with 80% of breast cancers, 70% of coloncancers, 90% of gynecological cancers, 50% of hepatocellular carcinomasand a variety of hematological tumors possessing abnormal Mycexpression. On the basis of these frequencies, it is estimated thatapproximately 100,000 U.S. cancer deaths per year are associated withchanges in the Myc gene or its expression. The clinical significance ofMyc gene alterations in human cancers is best illustrated byprognostication by N-Myc amplification in neuroblastoma, and the sinequa non chromosomal translocation affecting c-Myc in Burkitt's lymphoma.For a recent review of the Myc oncogene, see “Myc Oncogene,” TheEncyclopedia of Cancer, Second Edition, July 2002, Academic Press.

The c-Myc gene is located on human chromosome 8q24 and comprises threeexons. Transcription of the c-Myc gene can be initiated at one of threepromoters. Translation beginning at the AUG start site in the secondexon produces a major 439 amino acid protein, the 64 kDa c-Myc protein.Alternative translational initiation start sites result in both longerand shorter forms of the protein, termed p67 Myc and MycS, respectively.The c-Myc protein is O-glycosylated and phosphorylated, and thesemodifications can alter the protein half-life. The c-Myc sequencecontains several conserved N-terminal domains, termed Myc boxes, whichare found in closely related proteins, N-Myc and L-Myc.

The C-terminal region of c-Myc contains a dimerization motif, termed thehelix-loop-helix leucine zipper (HLH LZ). The HLH LZ domain mediateshomotypic or heterotypic dimerization with other HLH LZ proteins. Thec-Myc dimerization domain is important for cellular transformation, andthe bHLH LZ protein Max has been identified as a c-Myc obligate partnerprotein. The c-Myc RNA and protein have short half-lives as compared tothose of Max, and in most systems Myc appears to be the limiting,regulated component of the heterodimer. Dimerization of Myc and Maxthrough the HLH LZ domain aligns the adjacent basic regions on eachmolecule to grip onto specific DNA hexanucleotide core sequences, termedE boxes (5′-CA[C/T]GTG-3′). Max can also bind to bHLH LZ Mad familyproteins, which can mediate in transcriptional silencing. Mad levels, asopposed to those of Myc, increase during differentiation, and decreasedexpression of the Mad2 (Mxi-1) protein has been implicated in thedevelopment of cancer.

DNA-bound Myc-Max complexes activate transcription through the aminoterminal 143 amino acids of c-Myc, termed the transcriptional activationdomain (TAD). A small segment of this region is also important forMyc-mediated transcriptional repression. This N-terminal transregulatorydomain of c-Myc appears to be required for transformation, andtransformation appears to be highly dependent on the region required forMyc mediated transcriptional repression. Both of the activation andrepression functions of Myc may be required for neoplastictransformation.

The DNA bound Myc-Max heterodimer interacts through the Myc N-terminalregion with a variety of proteins involved in transcription. Theseinclude TRRAP, which associates with histone acetylase GCN5. Acetylationof histones may then mark chromatin to allow access of transcriptionfactors that belong to the general transcriptional machinery, such asTFIIE or TBP, to DNA. Other putative Myc binding proteins have beenimplicated in regulating Myc transactivating or transrepressingproperties, including p107 and Miz-1. The Mad-Max complexes, in contrastto Myc-Max complexes, recruit histone deacetylases that induce compactchromatin structures, which in turn can limit access of transcriptionfactors to DNA.

Expression of c-Myc in the normal cell is tightly regulated by externalsignals, such as growth factors and extracellular matrix contacts, aswell as by internal clocks, such as the cell cycle. The resting cellnormally expresses little c-Myc, whereas cells stimulated by growthfactors dramatically increase c-Myc expression such that c-Myc can betermed an immediate early response gene. c-Myc expression persists intothe cell cycle, but then returns to its basal quiescent state inresultant resting daughter cells. Abnormal or ectopic overexpression ofc-Myc in primary cells activates a protective pathway through theinduction of p19/p14ARF and a p53-dependent cell death pathway. Hence,normal cells that overexpress c-Myc are eliminated from the hostorganism through apoptosis, thereby protecting the organism from lethalneoplastic changes.

Normal embryonic development appears to require regulated expression ofc-Myc as well as other Myc family members. Mouse embryos in which bothalleles of c-Myc have been deleted by homologous recombination die inearly development with a lack of primitive hematopoiesis. Targeted genereplacement demonstrates that n-Myc can substitute for c-Myc duringembryogenesis, indicating that n-Myc functional domains are virtuallyidentical to those of c-Myc. L-Myc null animals have no distinctphenotype, suggesting that 1-Myc is not required for embryogenesis.Embryonic fibroblasts from animals lacking c-Myc show absent or a markeddecrease in proliferation rate and viability. The fruitfly Drosophilawith mutated Myc alleles are much smaller than wild-type flies, muchlike the Minute class of small fruitflies, which are mutant in ribosomalprotein genes and other yet-unidentified genes. The cells from thesemutant flies are small in size, indicating that Myc may participate inthe control of normal cell and organismal size. Murine hypomorphs ofc-Myc also cause smaller body size, but here it is due to the decreasein the number of cells rather than smaller cell size.

Activation of the c-Myc gene, which contributes to the development ofhuman cancers, occurs in several ways. Chromosomal translocations, as inthe case of Burkitt's lymphoma, activate the c-Myc locus by juxtaposingit adjacent to immunoglobulin genes that are transcriptionally highlyactive in B cells. Gene amplification increases Myc gene copy number,which in turn increases Myc expression. This is most dramatically foundin other members of the Myc family, where over 200 copies per cell ofN-Myc can be found in neuroblastoma, and over 50 copies per cell ofc-Myc, N-Myc, or L-Myc may be found in small cell lung cancers.Increased c-Myc gene transcription can account for the observedelevation of Myc in human colon carcinoma. Recent studies suggest thatthe transcriptional co-activator â-catenin, which is inactivated whenbound to the adenomatous polyposis coli (APC) protein, might activateMyc expression in the absence of APC through binding the TCF4transcription factor. Mutant, activated â-catenin proteins that arefound in colon, liver and other cancers may constitutively deregulateMyc expression leading to the development of these cancers.

Other mechanisms of c-Myc overexpression include removal of 3′ UTRdestabilizing sequences, resulting in an elevation of Myc mRNA.Insertion of retroviruses adjacent to the Myc locus activates itsexpression via retroviral regulatory sequences. Oncogenic ras appears tostabilize the Myc protein through a hypothesized post-translationalmodification mechanism.

A variety of models have been utilized to demonstrate that c-Myc isoncogenic. When immortalized Rat 1a fibroblasts are engineered tooverexpress c-Myc, they acquire the ability to grow in soft agar,signifying loss of contact inhibition commonly found in tumor cells.Furthermore, transgenic animals with tissue-targeted expression of c-Mycform tumors in the targeted tissues. More recently it has been shownthat the conditional induction of c-Myc transgene in vivo inkeratinocytes, mammary epithelial cells or hematopoietic cells can leadto reversible proliferation and clonal expansion, a hallmark ofneoplasia.

Although a link between c-Myc and cancer is well established both invivo and in vitro, recent work has established a role for c-Myc in cellcycle progression, metabolism, apoptosis and genomic instability. c-Mycis thought to promote cell proliferation and genomic instability byaccelerating cells through G1 and S phases of the cell cycle, abrogatingcell cycle checkpoints, and increasing cell metabolism. In many settingsthese alterations can lead to apoptosis, or cell death. However, in thebackground of additional mutations that activate anti-apoptotic signals,c-Myc can lead to complete neoplastic transformation.

Recent efforts have concentrated on identifying the genetic programinduced by c-Myc. Direct targets of c-Myc are defined as those targetgenes each of whose expression is directly altered by direct c-Mycbinding to each genes, respectively. Indirect targets are thoseactivated by genes that are direct targets of Myc (i.e. induced bytranscription factors, whose genes are direct c-Myc targets). c-Myctarget genes have been identified with several techniques that comparethe expression of genes in cell lines that overexpress c-Myc with onesthat do not. With the availability of cells that are depleted of c-Mycthrough homologous recombination, it is now more feasible to study therole of c-Myc in normal cell homeostasis. It is important, however, todistinguish between c-Myc targets induced in normal cell homeostasisversus those induced in pathophysiological, neoplastic conditions inwhich c-Myc is overexpressed. Myc target genes have been identified in avariety of ways, including by DNA microarray analysis and SAGE. Whilenone of the Myc responsive genes by itself recapitulates the completeMyc phenotype, many appear necessary and hence have been implicated inMyc functions, such as apoptosis, cell cycle control, and cell adhesion.

The removal of c-Myc by homologous recombination results in cells thathave remarkably slowed population growth doubling time. This slowedgrowth, however, appears to result in the decreased probability of acell to enter into the cell cycle in the absence of c-Myc. As a result,only a small fraction of the Myc null cell population enters the cellcycle with a virtually normal cell doubling time. This populationmultiplies for several cycles and then returns to a resting phase asother cell populations enter the cell cycle. These observations, whichare based on single cell studies, suggest that c-Myc controls theprobability of a cell to enter the cell cycle. The challenge is tounderstand how c-Myc affects the stochastic nature of cell proliferationat the molecular level. Most studies have focused on c-Myc's effect onregulatory proteins of the G1-S phase transition of the cell cycle. Thistransition is promoted when cyclin dependent kinases (CDKs) areactivated by association with specific cyclins. Cyclin dependent kinaseinhibitors (CDKIs) inhibit this activation. c-Myc has been implicated ininducing cyclin D1 and D2, cyclin E, CDK4, and cdc25A, a phosphatasewhich activates CDK2 and CDK4. c-Myc has also been shown to lower theamounts or inhibit the function of the CDK inhibitor, p27, potentiallyby increasing cyclin D levels which can then sequester p27. c-Myc alsoinduces Cull, which mediates the degradation of p27.

A highly regulated cell cycle permits cells to repair DNA damage beforereplicating, thus promoting genomic fidelity. Inappropriate cell cycleproliferation can lead to genomic instability, resulting in newmutations and abnormal chromosome number and structure. c-Mycoverexpression, even transiently, can induce genomic instability that ischaracterized by gene amplification, aneuploidy and polyploidy. Otherstudies suggest that c-Myc induces the production of reactive oxygenspecies (ROS) by mitochondria, leading to DNA damage and genomicinstability.

Burkitt's lymphoma has long been noted by pathologists to have not onlya high mitotic index, but also by a high apoptotic rate as illustratedby the abundance of abnormal nuclei. c-Myc induced apoptosis was firstrecognized in studies of the IL-3 dependent 32d.3 myeloid progenitorcell line. In the absence of IL-3, enforced c-Myc expression drivescells into S phase and accelerates the rate of cell death. Fibroblaststhat overexpress c-Myc also undergo apoptosis in response toenvironmental stresses, including low serum, hypoxia, and low glucose.The regions of Myc that are required for apoptosis are also those thatare required for cellular transformation. Although several Myc targetgenes, such as ornithine decarboxylase (ODC) and lactate dehydrogenase A(LDH-A), appear to be involved in Myc-induced apoptosis, the mechanismof Myc action in apoptosis is not clear. In the case of primary cells,p19/p14ARF is likely to be a relevant downstream effector of Myc-inducedapoptosis. Myc also activates the telomerase hTERT, which encodes anenzyme that sustains telomere length and leads to immortalization ofcells. The relevant clinical correlation is the intriguing observationthat both N-Myc amplification and telomerase levels are parallelpredictors of poor outcome in neuroblastoma. This observation can beconstrued to suggest that N-Myc is responsible for the elevation oftelomerase in neuroblastoma.

Proliferation requires increased energy and availability of biosyntheticsubstrates, and would therefore necessitate increase metabolic rates. Itis well established that the major component of a eukaryotic cell is itsribosomal mass, which constitutes up to 40% of total mass and accountsfor 90% of cellular RNA mass. It is also notable that as a celltraverses through the G1 phase of the cell cycle, its mass graduallyincreases prior to the initiation of DNA synthesis in S phase. Theregulation of cell mass is linked to the cell cycle, although themolecular basis for this regulation is largely unknown. Intriguingly,mutation of the Drosophila Myc gene results in small flies that resembleflies that bear mutant ribosomal protein genes. These small flies aresmall not because they have fewer cells, but rather their cells aresmaller in size. These observations suggest that Myc participates in theregulation of cell mass, and this notion has gained support fromobservations in specific mammalian cells that overexpress c-Myc. Recentstudies have suggested that c-Myc overexpression results in elevatedexpression of genes encoding translational factors and ribosomalproteins, which in turn may contribute to increased cellular mass.

Both large and small tumors are often hypoxic as a result of diminished,disordered neovascularization. Survival and rapid proliferation of atumor in a hostile environment is often accompanied by a switch ofcancer cells to use glucose as an energy source without oxygen and leadsto an overproduction of lactic acid. The transcription factor, HIF-1, isinduced by hypoxia and transactivates many glycolytic enzymes necessaryfor this switch. Interestingly, HIF-1 binds to a DNA motif in thepromoter of these genes that is very similar to the Myc E-box. Myc isable to transactivate many of these same glycolytic genes and theglucose transporter GLUT-1. Hence, the widespread deregulation of c-Mycin human cancers may contribute to enhanced tumor glycolysis, known asthe Warburg effect. Recently, c-Myc was shown to regulate a nuclear geneencoding a mitochondrial peroxiredoxin, which appears required for cellproliferation and transformation mediated by c-Myc. Furthermore, themitochondrial serine hydroxymethyltransferase, which is involved insingle carbon metabolism, was able to partially rescue the growth of ratMyc null cells in an expression cloning strategy. Hence, the pleiotropiceffects of c-Myc on cellular metabolism and cell growth remain to beclearly delineated.

As cells prepare for S phase, adequate substrates must accumulate toensure the fidelity of DNA replication. Genes that are important innucleotide biosynthesis and DNA metabolism are also implicated as Myctargets, such as carbamoyl phosphate synthase (CAD), ODC, dihydrofolatereductase and thymidine kinase. Although little is known about thepossible role of c-Myc in regulating cellular metabolism during thesecond cell cycle gap, G2, and during mitosis, a complex role for c-Mycin regulating cellular metabolism has clearly emerged from recentstudies.

Based upon the current understanding of Myc biology and biochemistry,the modulation of c-Myc and other genes involved in the Myc pathway isinstrumental in the development of new therapeutics in the field ofoncology, including in related fields of cell cycle regulation,apoptosis, metabolism, cell adhesion, and cell differentiation. As such,modulation of Myc genes and other genes involved in the Myc pathwayusing small interfering nucleic acid (siNA) mediated RNAi represents anovel approach to the treatment, diagnosis, and study of diseases andconditions related to Myc activity and/or gene expression, includingcancer and other proliferative disorders, such as restenosis.

Myb Biology and Biochemistry

The c-myb proto-oncogene is the cellular homolog of the avianmyeloblastosis virus transforming gene v-myb. The c-myb protein is atranscription factor involved in the regulation of hematopoietic cellproliferation and differentiation. C-myb is highly expressed inprimitive hematopoietic cells, and its expression decreases duringmaturation and differentiation of hematopoietic cells. C-myb expressionis also elevated in some primary hematopoietic tumors and in someleukemic cell lines. The in vitro and in vivo expression of c-myb canresult in transformation of preferentially myeloid lineage hematopoieticcells. Since the myb gene encodes proteins that are critical forhematopoietic cell proliferation and development, disrupting mybfunction has been proposed as an effective therapeutic strategy forcontrolling leukemic cell growth. An animal model for testing the invivo efficacy of antisense oligodeoxynucleotides targeting myb mRNA hasbeen developed (Ratajczak et al., 1992, Proc. Nat. Acad. Sci., 89,11823-11827). This model comprises a human leukemiascid mouse chimerawith K562 cells derived from a patient with chronic myelogenousleukemia. Mice treated with the antisense targeting myb expressionsurvived at least 3.5 times longer than control animals. In addition,animals receiving antisense myb DNA had significantly less disease atthe two sites most frequently involved by leukemic cell infiltration,namely the CNS and the ovary.

The myb gene is also implicated in restenosis following angioplasty.When whole serum is added to serum-starved smooth muscle cells in vitro,the oncogenes c-myc, c-fos, and c-myb, are induced (Kindy andSonenshein, 1986, J. Biol. Chem., 261, 12865-12868) and cellproliferation ensues. Blocking c-myb with an antisense oligonucleotideprevents cells from entering S phase (Brown, K. E., et al., 1992, J.Biol. Chem., 267, 4625-4630). Thus, c-myb is thought to be required forthe G₁ to S transition after stimulation by the multitude of growthfactors present in serum. In vivo, a c-myb antisense oligonucleotideinhibits restenosis when applied to rat arteries after balloonangioplasty (Simons, M., et al., 1992, Nature, 359, 67-70). Similarly,an antisense oligonucleotide directed against mRNA of the oncogene c-Mycwas shown to inhibit human smooth muscle cell proliferation (Shi, Y., etal., 1993, Circulation, 88, 1190-5) and migration (Biro, S., et al.,1993, Proc. Natl. Acad. Sci. U S A, 90, 654-8).

Therefore, the use of small interfering nucleic acid molecules targetingcellular oncogenes, such as c-myb, and genes involved in the c-mybexpression pathway, such as IL-2, bcl-2, protein kinase B (PKB),phosphoinositide 3-kinase (PI3K), E2F, NFkappaB, c-myc, and pim-1,provides a class of novel therapeutic agents that can be used in thetreatment of various cancers, such as leukemia and proliferativeconditions such as restenosis.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H20 followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi: 10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a Mycand/or Myb target sequence is used to screen for target sites in cellsexpressing Myc and/or Myb RNA, such as cultured human aortic smoothmuscle cells (e.g., HASMC) for Myb RNA and HeLa cells for Myc RNA. Thegeneral strategy used in this approach is shown in FIG. 9. Anon-limiting example of such is a pool comprising sequences having anyof SEQ ID NOs 1-651 and 656-682. Cells expressing Myc and/or Myb (e.g.,cultured human aortic smooth muscle cells (e.g., HASMC) for Myb RNA andHeLa cells for Myc RNA) are transfected with the pool of siNA constructsand cells that demonstrate a phenotype associated with Myc and/or Mybinhibition are sorted. The pool of siNA constructs can be expressed fromtranscription cassettes inserted into appropriate vectors (see forexample FIG. 7 and FIG. 8). The siNA from cells demonstrating a positivephenotypic change (e.g., decreased proliferation, decreased Myc and/orMyb mRNA levels or decreased Myc and/or Myb protein expression), aresequenced to determine the most suitable target site(s) within thetarget Myc and/or Myb RNA sequence.

Example 4 Myc and/or Myb Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the Myc and/orMyb RNA target and optionally prioritizing the target sites on the basisof folding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically-modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi in Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting Myc and/or Myb RNA targets. Theassay comprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with Myc and/or Myb target RNA. A Drosophila extractderived from syncytial blastoderm is used to reconstitute RNAi activityin vitro. Target RNA is generated via in vitro transcription from anappropriate Myc and/or Myb expressing plasmid using T7 RNA polymerase orvia chemical synthesis as described herein. Sense and antisense siNAstrands (for example 20 uM each) are annealed by incubation in buffer(such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mMmagnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C.,then diluted in lysis buffer (for example 100 mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can bemonitored by gel electrophoresis on an agarose gel in TBE buffer andstained with ethidium bromide. The Drosophila lysate is prepared usingzero to two-hour-old embryos from Oregon R flies collected on yeastedmolasses agar that are dechlorinated and lysed. The lysate iscentrifuged and the supernatant isolated. The assay comprises a reactionmixture containing 50% lysate [vol/vol], RNA (10-50 pM finalconcentration), and 10% [vol/vol] lysis buffer containing siNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in theMyc and/or Myb RNA target for siNA mediated RNAi cleavage, wherein aplurality of siNA constructs are screened for RNAi mediated cleavage ofthe Myc and/or Myb RNA target, for example, by analyzing the assayreaction by electrophoresis of labeled target RNA, or by Northernblotting, as well as by other methodology well known in the art.

Example 7 Nucleic Acid Inhibition of Myc and/or Myb Target RNA

siNA molecules targeted to the human Myc and/or Myb RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theMyc and/or Myb RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting Myc and/orMyb. First, the reagents are tested in cell culture using, for example,cultured HeLa or HASMC cells, to determine the extent of RNA and proteininhibition. siNA reagents (e.g.; see Tables II and III) are selectedagainst the Myc and/or Myb target as described herein. RNA inhibition ismeasured after delivery of these reagents by a suitable transfectionagent to, for example, HeLa or HASMC cells. Relative amounts of targetRNA are measured versus actin using real-time PCR monitoring ofamplification (e.g., ABI 7700 TAQMAN®). A comparison is made to amixture of oligonucleotide sequences made to unrelated targets or to arandomized siNA control with the same overall length and chemistry, butrandomly substituted at each position. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., HeLa or HASMC cells) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaker) the day beforetransfection. siNA (final concentration, for example 20 nM) and cationiclipid (e.g., final concentration 2 μg/ml) are complexed in EGM basalmedia (Bio Whittaker) at 37° C. for 30 minutes in polystyrene tubes.Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTF, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of Myc GeneExpression Cell Culture

There are numerous cell culture systems that can be used to analyzereduction of Myc levels either directly or indirectly by measuringdownstream effects. For example, cultured HeLa cells can be used in cellculture experiments to assess the efficacy of nucleic acid molecules ofthe invention. As such, HeLa cells treated with nucleic acid moleculesof the invention (e.g., siNA) targeting Myc RNA would be expected tohave decreased Myc expression capacity compared to matched controlnucleic acid molecules having a scrambled or inactive sequence. In anon-limiting example, HeLa cells are cultured and Myc expression isquantified, for example by time-resolved immuno fluorometric assay. Mycmessenger-RNA expression is quantitated with RT-PCR in cultured HeLas.Untreated cells are compared to cells treated with siNA moleculestransfected with a suitable reagent, for example a cationic lipid suchas lipofectamine, and Myc protein and RNA levels are quantitated. Doseresponse assays are then performed to establish dose dependentinhibition of Myc expression.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-Myc agents in animal models is animportant prerequisite to human clinical trials. Barisoni et al., 1995,Am J. Pathol., 147, 1728-35 describe a transgenic mouse model ofpolycystic kidney disease. Adult polycystic kidney disease is believedto be the most frequent (1/500) inherited genetic disorder in humans.Barisoni et al., supra generated a genetic model of the disease intransgenic mice by introducing a deregulated proto-oncogene c-mycspecifically expressed in the kidney. All transgenic lines produceddevelop adult polycystic kidney disease in a reproducible manner. Theclinical phenotype observed in mice is present at birth and leads torenal insufficiency in adulthood. The investigators determined thatabnormal proliferation and programmed cell death are responsible forcystogenesis in polycystic kidney disease. Furthermore, this phenomenais controlled by a specific c-myc mechanism independent of the p53pathway. A similar mechanism also prevails in human autosomal dominantpolycystic kidney disease. Therefore, this murine model provides auseful model to understand the polycystic kidney disease pathogenesisand can be used to evaluate potential therapeutic agents such as siNAmolecules of the invention.

Other animal models known in the art can be used to evaluate siNAmolecules of the invention targeting c-Myc for other disease conditions,see for example Schmidt et al., 1988. PNAS USA., 85:6047-6051(describing transgenic mice bearing the human c-myc gene activated by animmunoglobulin enhancer useful as a pre-B-cell lymphoma model) and Hoyeret al., 2002, PNAS USA., 99, 14392-97 (describing transgenic mice thatdevelop Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma(DLBCL)). Animals treated with siNA molecules of the invention targetingMyc RNA can be evaluated for clinical response (e.g., decreased tumorsize/metastasis) and/or decreased levels of Myc RNA or protein.

Example 9 Models Useful to Evaluate the Down-Regulation of Myb GeneExpression Cell Culture

Rat vascular smooth muscle cells (RASMC) are an example of cells thatcan be used to analyze reduction of Myb levels either directly orindirectly by measuring downstream effects. Rat vascular smooth musclecells are isolated and cultured as follows. Aortas from adultSprague-Dawley rats are dissected, connective tissue is removed under adissecting microscope, and 1 mm² pieces of the vessel are placed,intimal side up, in a Petri dish in Modified Eagle's Medium (MEM) withthe following additives: 10% FBS, 2% tryptose phosphate broth, 1%penicillin/streptomycin and 2 mM L-Glutamine. The smooth muscle cellsare allowed to migrate and grow to confluence over a 3-4 week period.These primary cells are frozen and subsequent passages are grown at 37°C. in 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM), 10% fetalbovine serum (FBS), and the following additives: 2 mM L-Glutamine, 1%penicillin/streptomycin, 1 mM sodium pyruvate, non-essential amino acids(0.1 mM of each amino acid), and 20 mM Hepes pH 7.4. Cells passed fourto six times are used in proliferation assays. For the cellproliferation assays, 24-well tissue culture plates are prepared bycoating the wells with 0.2% gelatin and washing once withphosphate-buffered saline (PBS). RASMC are inoculated at 1×10⁴ cells perwell in 1 ml of DMEM plus 10% FBS and additives and incubated for 24hours. The cells become sub confluent when plated at this density. Thecells are serum-starved by removing the medium, washing once with PBS,and incubating 48-72 hours in DMEM containing 0.5% FBS plus additives.

In several other systems, cationic lipids have been shown to enhance thebioavailability of oligonucleotides to cells in culture (Bennet, et al.,1992, Mol. Pharmacology, 41, 1023-1033). In one embodiment, siNAmolecules of the invention are complexed with cationic lipids for cellculture experiments. siNA and cationic lipid mixtures are prepared inserum-free DMEM immediately prior to addition to the cells. DMEM plusadditives are warmed to room temperature (about 20-25° C.) and cationiclipid is added to the final desired concentration and the solution isvortexed briefly. siNA molecules are added to the final desiredconcentration and the solution is again vortexed briefly and incubatedfor 10 minutes at room temperature. In dose response experiments, theRNA/lipid complex is serially diluted into DMEM following the 10 minuteincubation.

Serum-starved smooth muscle cells are washed twice with PBS, and thesiNA/lipid complex is added. The plates are incubated for 4 hours at 37°C. The medium is then removed and DMEM containing 10% FBS, additives and10 μM bromodeoxyuridine (BrdU) is added. In some wells, FBS is omittedto determine the baseline of unstimulated proliferation. The plates areincubated at 37° C. for 20-24 hours, fixed with 0.3% hydrogen peroxidein 100% methanol, and stained for BrdU incorporation by standardmethods. In this procedure, cells that have proliferated andincorporated BrdU stain brown; non-proliferating cells arecounter-stained a light purple. Both BrdU positive and BrdU negativecells are counted under the microscope. The percentage of the totalcells that have incorporated BrdU (% cell proliferation) is determined.Percent inhibition is calculated from the % cell proliferation values asfollows: % inhibition=100−100[(siNA−0% serum)/(Control−0% serum)]. Assuch, the efficacy of siNA molecules of the invention targeting Myb canbe determined via inhibition of smooth muscle cell proliferation.

Animal Models

Evaluating the efficacy of anti-Myb agents in animal models is animportant prerequisite to human clinical trials. Smooth muscle cell(SMC) proliferation is an important component of restenosis in responseto injury after balloon angioplasty. The rat carotid artery ballooninjury model is a well-characterized and highly reproducible vascularproliferative disorder that is dependent on SMC migration andproliferation. Accordingly, this model can be used to determine whetherthe inhibition of SMC proliferation by siNA observed in cell cultureexperiments is sufficient to impact neointima formation in a rat carotidartery model of balloon angioplasty. Rat carotid arteries are subjectedto balloon angioplasty and immediately exposed to siNA or inactivecontrol siNA molecules. In a non-limiting example, Sprague-Dawley ratsare subjected to balloon angioplasty of the left common carotid arteryby dilatation with a Fogarty catheter. Immediately following injury,siNA, or inactive control siNA molecules in a volume of 50 to 100 μl isinstilled into a 1-cm segment of the distal common carotid artery for 5minutes by using a 24-gauge intravenous catheter. Rat carotid arteriesare harvested 20 days after balloon injury and adenovirus infection.Tissue sections are stained with hematoxylin and eosin. Intimal andmedial boundaries are determined by digital planimetry of tissuesections. Areas and ratios are determined from four to six stainedsections of each artery spanning the 1-cm site of balloon injury. Assuch, the efficacy of siNA molecules of the invention can be determinedvia inhibition of smooth muscle cell proliferation in the rat carotidartery balloon injury model.

Example 10 RNAi Mediated Inhibition of Myc Expression in Cell Culture

Inhibition of Myc RNA Expression Using siNA Targeting Myc RNA

siNA constructs (e.g., Table III) are tested for efficacy in reducingMyc (c-Myc) RNA expression in, for example, 293T cells. Cells are platedapproximately 24 hours before transfection in 96-well plates at5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes atroom temperature. The siNA transfection mixtures are added to cells togive a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the target gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data areaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

Results of this study are shown in FIG. 23. A screen of siNA constructswas compared to untreated cells, scrambled siNA control constructs(Scram1 and Scram2), and cells transfected with lipid alone(transfection control). As shown in the figure, three of the siNAconstructs (Compound number 30993/31069; Compound number 30995/31071;and Compound number 30996/31072) show significant reduction of c-Myc RNAexpression. Additional stabilization chemistries as described in TableIV are similarly assayed for activity.

Example 11 Indications

The present body of knowledge in Myc and Myb research indicates the needfor methods and compounds that can regulate Myc and/or Myb gene productexpression for research, diagnostic, and therapeutic use. As describedherein, the nucleic acid molecules of the present invention can be usedto treat leukemias including acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia; ovarian cancer, breast cancer, cancers ofthe head and neck, lymphomas such as mantle cell lymphoma, non-Hodgkin'slymphoma, and Burkitt's lymphoma, adenoma, squamous cell carcinoma,laryngeal carcinoma, multiple myeloma, melanoma, colorectal cancer,prostate cancer, and proliferative diseases such as restenosis,polycystic kidney disease and any other diseases or conditions that arerelated to or will respond to the levels of Myc and/or Myb in a cell ortissue, alone or in combination with other therapies.

The use of radiation treatments and chemotherapeutics, such asGemcytabine and cyclophosphamide, are non-limiting examples ofchemotherapeutic agents that can be combined with or used in conjunctionwith the nucleic acid molecules (e.g., siNA molecules) of the instantinvention. Those skilled in the art will recognize that otheranti-cancer and/or antiproliferative compounds and therapies can besimilarly be readily combined with the nucleic acid molecules of theinstant invention (e.g., siNA molecules) and are hence within the scopeof the instant invention. Such compounds and therapies are well known inthe art (see for example Cancer: Principles and Practice of Oncology,Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A.,J. B. Lippincott Company, Philadelphia, USA; incorporated herein byreference) and include, without limitations, folates, antifolates,pyrimidine analogs, fluoropyrimidines, purine analogs, adenosineanalogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids,antibiotics, anthacyclins, platinum analogs, alkylating agents,nitrosoureas, plant derived compounds such as vinca alkaloids,epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiationtherapy, surgery, nutritional supplements, gene therapy, radiotherapy,for example 3D-CRT, immunotoxin therapy, for example ricin, andmonoclonal antibodies. Specific examples of chemotherapeutic compoundsthat can be combined with or used in conjunction with the nucleic acidmolecules of the invention include, but are not limited to, Paclitaxel;Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tamoxifen;Leucovorin; 5-fluoro uridine (5-FU); Ionotecan; Cisplatin; Carboplatin;Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin;Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogenmustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide;4-hydroperoxycyclophosphamide, Thiotepa; Irinotecan (CAMPTOSAR®, CPT-11,Camptothecin-11, Campto) Tamoxifen, Herceptin; IMC C225; ABX-EGF: andcombinations thereof. The above list provides non-limiting examples ofcompounds and/or methods that can be combined with or used inconjunction with the nucleic acid molecules (e.g., siNA) of the instantinvention. Those skilled in the art will recognize that other drugcompounds and therapies can be similarly be readily combined with thenucleic acid molecules of the instant invention (e.g., siNA molecules)are hence within the scope of the instant invention.

Example 12 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Myc and Myb Accession Numbers Myc Accession Numbers MYCNM_002467 Homo sapiens v-myc myelocytomatosis (C-myc, V-myc) viraloncogene homolog (avian) (MYC), mRNA MYCL1 (L-myc) NM_005376 Homosapiens v-myc myelocytomatosis viral oncogene homolog 1, lung carcinomaderived (avian) (MYCL1), mRNA MYCL2 (L-myc) NM_005377 Homo sapiens v-mycmyelocytomatosis viral oncogene homolog 2 (avian) (MYCL2), mRNA MYCN(N-myc) NM_005378 Homo sapiens v-myc myelocytomatosis viral relatedoncogene, neuroblastoma derived (avian) (MYCN), mRNA Myb AccessionNumbers MYB NM_005375 Homo sapiens v-myb myeloblastosis (C-myb, V-myb)viral oncogene homolog (avian) (MYB), mRNA MYBL1 (A-myb) X66087 H.sapiens a-myb mRNA MYBL2 (B-myb) NM_002466 Homo sapiens v-mybmyeloblastosis viral oncogene homolog (avian)-like 2 (MYBL2), mRNA

TABLE II Myc and Myb siNA and Target Sequences Pos Target Sequence SeqID UPos Upper seq Seq ID LPos Lower seq Seq ID NM 002467 (hMYC)    1CUGCUCGCGGCCGCCACCG   1    1 CUGCUCGCGGCCGCCACCG   1   19CGGUGGCGGCCGCGAGCAG 119   19 GCCGGGCCCCGGCCGUCCC   2   19GCCGGGCCCCGGCCGUCCC   2   37 GGGACGGCCGGGGCCCGGC 120   37CUGGCUCCCCUCCUGCCUC   3   37 CUGGCUCCCCUCCUGCCUC   3   55GAGGCAGGAGGGGAGCCAG 121   55 CGAGAAGGGCAGGGCUUCU   4   55CGAGAAGGGCAGGGCUUCU   4   73 AGAAGCCCUGCCCUUCUCG 122   73UCAGAGGCUUGGCGGGAAA   5   73 UCAGAGGCUUGGCGGGAAA   5   91UUUCCCGCCAAGCCUCUGA 123   91 AAAAGAACGGAGGGAGGGA   6   91AAAAGAACGGAGGGAGGGA   6  109 UCCCUCCCUCCGUUCUUUU 124  109AUCGCGCUGAGUAUAAAAG   7  109 AUCGCGCUGAGUAUAAAAG   7  127CUUUUAUACUCAGCGCGAU 125  127 GCCGGUUUUCGGGGCUUUA   8  127GCCGGUUUUCGGGGCUUUA   8  145 UAAAGCCCCGAAAACCGGC 126  145AUCUAACUCGCUGUAGUAA   9  145 AUCUAACUCGCUGUAGUAA   9  163UUACUACAGCGAGUUAGAU 127  163 AUUCCAGCGAGAGGCAGAG  10  163AUUCCAGCGAGAGGCAGAG  10  181 CUCUGCCUCUCGCUGGAAU 128  181GGGAGCGAGCGGGCGGCCG  11  181 GGGAGCGAGCGGGCGGCCG  11  199CGGCCGCCCGCUCGCUCCC 129  199 GGCUAGGGUGGAAGAGCCG  12  199GGCUAGGGUGGAAGAGCCG  12  217 CGGCUCUUCCACCCUAGCC 130  217GGGCGAGCAGAGCUGCGCU  13  217 GGGCGAGCAGAGCUGCGCU  13  235AGCGCAGCUCUGCUCGCCC 131  235 UGCGGGCGUCCUGGGAAGG  14  235UGCGGGCGUCCUGGGAAGG  14  253 CCUUCCCAGGACGCCCGCA 132  253GGAGAUCCGGAGCGAAUAG  15  253 GGAGAUCCGGAGCGAAUAG  15  271CUAUUCGCUCCGGAUCUCC 133  271 GGGGGCUUCGCCUCUGGCC  16  271GGGGGCUUCGCCUCUGGCC  16  289 GGCCAGAGGCGAAGCCCCC 134  289CCAGCCCUCCCGCUUGAUC  17  289 CCAGCCCUCCCGCUUGAUC  17  307GAUCAAGCGGGAGGGCUGG 135  307 CCCCCAGGCCAGCGGUCCG  18  307CCCCCAGGCCAGCGGUCCG  18  325 CGGACCGCUGGCCUGGGGG 136  325GCAACCCUUGCCGCAUCCA  19  325 GCAACCCUUGCCGCAUCCA  19  343UGGAUGCGGCAAGGGUUGC 137  343 ACGAAACUUUGCCCAUAGC  20  343ACGAAACUUUGCCCAUAGC  20  361 GCUAUGGGCAAAGUUUCGU 138  361CAGCGGGCGGGCACUUUGC  21  361 CAGCGGGCGGGCACUUUGC  21  379GCAAAGUGCCCGCCCGCUG 139  379 CACUGGAACUUACAACACC  22  379CACUGGAACUUACAACACC  22  397 GGUGUUGUAAGUUCCAGUG 140  397CCGAGCAAGGACGCGACUC  23  397 CCGAGCAAGGACGCGACUC  23  415GAGUCGCGUCCUUGCUCGG 141  415 CUCCCGACGCGGGGAGGCU  24  415CUCCCGACGCGGGGAGGCU  24  433 AGCCUCCCCGCGUCGGGAG 142  433UAUUCUGCCCAUUUGGGGA  25  433 UAUUCUGCCCAUUUGGGGA  25  451UCCCCAAAUGGGCAGAAUA 143  451 ACACUUCCCCGCCGCUGCC  26  451ACACUUCCCCGCCGCUGCC  26  469 GGCAGCGGCGGGGAAGUGU 144  469CAGGACCCGCUUCUCUGAA  27  469 CAGGACCCGCUUCUCUGAA  27  487UUCAGAGAAGCGGGUCCUG 145  487 AAGGCUCUCCUUGCAGCUG  28  487AAGGCUCUCCUUGCAGCUG  28  505 CAGCUGCAAGGAGAGCCUU 146  505GCUUAGACGCUGGAUUUUU  29  505 GCUUAGACGCUGGAUUUUU  29  523AAAAAUCCAGCGUCUAAGC 147  523 UUUCGGGUAGUGGAAAACC  30  523UUUCGGGUAGUGGAAAACC  30  541 GGUUUUCCACUACCCGAAA 148  541CAGCAGCCUCCCGCGACGA  31  541 CAGCAGCCUCCCGCGACGA  31  559UCGUCGCGGGAGGCUGCUG 149  559 AUGCCCCUCAACGUUAGCU  32  559AUGCCCCUCAACGUUAGCU  32  577 AGCUAACGUUGAGGGGCAU 150  577UUCACCAACAGGAACUAUG  33  577 UUCACCAACAGGAACUAUG  33  595CAUAGUUCCUGUUGGUGAA 151  595 GACCUCGACUACGACUCGG  34  595GACCUCGACUACGACUCGG  34  613 CCGAGUCGUAGUCGAGGUC 152  613GUGCAGCCGUAUUUCUACU  35  613 GUGCAGCCGUAUUUCUACU  35  631AGUAGAAAUACGGCUGCAC 153  631 UGCGACGAGGAGGAGAACU  36  631UGCGACGAGGAGGAGAACU  36  649 AGUUCUCCUCCUCGUCGCA 154  649UUCUACCAGCAGCAGCAGC  37  649 UUCUACCAGCAGCAGCAGC  37  667GCUGCUGCUGCUGGUAGAA 155  667 CAGAGCGAGCUGCAGCCCC  38  667CAGAGCGAGCUGCAGCCCC  38  685 GGGGCUGCAGCUCGCUCUG 156  685CCGGCGCCCAGCGAGGAUA  39  685 CCGGCGCCCAGCGAGGAUA  39  703UAUCCUCGCUGGGCGCCGG 157  703 AUCUGGAAGAAAUUCGAGC  40  703AUCUGGAAGAAAUUCGAGC  40  721 GCUCGAAUUUCUUCCAGAU 158  721CUGCUGCCCACCCCGCCCC  41  721 CUGCUGCCCACCCCGCCCC  41  739GGGGCGGGGUGGGCAGCAG 159  739 CUGUCCCCUAGCCGCCGCU  42  739CUGUCCCCUAGCCGCCGCU  42  757 AGCGGCGGCUAGGGGACAG 160  757UCCGGGCUCUGCUCGCCCU  43  757 UCCGGGCUCUGCUCGCCCU  43  775AGGGCGAGCAGAGCCCGGA 161  775 UCCUACGUUGCGGUCACAC  44  775UCCUACGUUGCGGUCACAC  44  793 GUGUGACCGCAACGUAGGA 162  793CCCUUCUCCCUUCGGGGAG  45  793 CCCUUCUCCCUUCGGGGAG  45  811CUCCCCGAAGGGAGAAGGG 163  811 GACAACGACGGCGGUGGCG  46  811GACAACGACGGCGGUGGCG  46  829 CGCCACCGCCGUCGUUGUC 164  829GGGAGCUUCUCCACGGCCG  47  829 GGGAGCUUCUCCACGGCCG  47  847CGGCCGUGGAGAAGCUCCC 165  847 GACCAGCUGGAGAUGGUGA  48  847GACCAGCUGGAGAUGGUGA  48  865 UCACCAUCUCCAGCUGGUC 166  865ACCGAGCUGCUGGGAGGAG  49  865 ACCGAGCUGCUGGGAGGAG  49  883CUCCUCCCAGCAGCUCGGU 167  883 GACAUGGUGAACCAGAGUU  50  883GACAUGGUGAACCAGAGUU  50  901 AACUCUGGUUCACCAUGUC 168  901UUCAUCUGCGACCCGGACG  51  901 UUCAUCUGCGACCCGGACG  51  919CGUCCGGGUCGCAGAUGAA 169  919 GACGAGACCUUCAUCAAAA  52  919GACGAGACCUUCAUCAAAA  52  937 UUUUGAUGAAGGUCUCGUC 170  937AACAUCAUCAUCCAGGACU  53  937 AACAUCAUCAUCCAGGACU  53  955AGUCCUGGAUGAUGAUGUU 171  955 UGUAUGUGGAGCGGCUUCU  54  955UGUAUGUGGAGCGGCUUCU  54  973 AGAAGCCGCUCCACAUACA 172  973UCGGCCGCCGCCAAGCUCG  55  973 UCGGCCGCCGCCAAGCUCG  55  991CGAGCUUGGCGGCGGCCGA 173  991 GUCUCAGAGAAGCUGGCCU  56  991GUCUCAGAGAAGCUGGCCU  56 1009 AGGCCAGCUUCUCUGAGAC 174 1009UCCUACCAGGCUGCGCGCA  57 1009 UCCUACCAGGCUGCGCGCA  57 1027UGCGCGCAGCCUGGUAGGA 175 1027 AAAGACAGCGGCAGCCCGA  58 1027AAAGACAGCGGCAGCCCGA  58 1045 UCGGGCUGCCGCUGUCUUU 176 1045AACCCCGCCCGCGGCCACA  59 1045 AACCCCGCCCGCGGCCACA  59 1063UGUGGCCGCGGGCGGGGUU 177 1063 AGCGUCUGCUCCACCUCCA  60 1063AGCGUCUGCUCCACCUCCA  60 1081 UGGAGGUGGAGCAGACGCU 178 1081AGCUUGUACCUGCAGGAUC  61 1081 AGCUUGUACCUGCAGGAUC  61 1099GAUCCUGCAGGUACAAGCU 179 1099 CUGAGCGCCGCCGCCUCAG  62 1099CUGAGCGCCGCCGCCUCAG  62 1117 CUGAGGCGGCGGCGCUCAG 180 1117GAGUGCAUCGACCCCUCGG  63 1117 GAGUGCAUCGACCCCUCGG  63 1135CCGAGGGGUCGAUGCACUC 181 1135 GUGGUCUUCCCCUACCCUC  64 1135GUGGUCUUCCCCUACCCUC  64 1153 GAGGGUAGGGGAAGACCAC 182 1153CUCAACGACAGCAGCUCGC  65 1153 CUCAACGACAGCAGCUCGC  65 1171GCGAGCUGCUGUCGUUGAG 183 1171 CCCAAGUCCUGCGCCUCGC  66 1171CCCAAGUCCUGCGCCUCGC  66 1189 GCGAGGCGCAGGACUUGGG 184 1189CAAGACUCCAGCGCCUUCU  67 1189 CAAGACUCCAGCGCCUUCU  67 1207AGAAGGCGCUGGAGUCUUG 185 1207 UCUCCGUCCUCGGAUUCUC  68 1207UCUCCGUCCUCGGAUUCUC  68 1225 GAGAAUCCGAGGACGGAGA 186 1225CUGCUCUCCUCGACGGAGU  69 1225 CUGCUCUCCUCGACGGAGU  69 1243ACUCCGUCGAGGAGAGCAG 187 1243 UCCUCCCCGCAGGGCAGCC  70 1243UCCUCCCCGCAGGGCAGCC  70 1261 GGCUGCCCUGCGGGGAGGA 188 1261CCCGAGCCCCUGGUGCUCC  71 1261 CCCGAGCCCCUGGUGCUCC  71 1279GGAGCACCAGGGGCUCGGG 189 1279 CAUGAGGAGACACCGCCCA  72 1279CAUGAGGAGACACCGCCCA  72 1297 UGGGCGGUGUCUCCUCAUG 190 1297ACCACCAGCAGCGACUCUG  73 1297 ACCACCAGCAGCGACUCUG  73 1315CAGAGUCGCUGCUGGUGGU 191 1315 GAGGAGGAACAAGAAGAUG  74 1315GAGGAGGAACAAGAAGAUG  74 1333 CAUCUUCUUGUUCCUCCUC 192 1333GAGGAAGAAAUCGAUGUUG  75 1333 GAGGAAGAAAUCGAUGUUG  75 1351CAACAUCGAUUUCUUCCUC 193 1351 GUUUCUGUGGAAAAGAGGC  76 1351GUUUCUGUGGAAAAGAGGC  76 1369 GCCUCUUUUCCACAGAAAC 194 1369CAGGCUCCUGGCAAAAGGU  77 1369 CAGGCUCCUGGCAAAAGGU  77 1387ACCUUUUGCCAGGAGCCUG 195 1387 UCAGAGUCUGGAUCACCUU  78 1387UCAGAGUCUGGAUCACCUU  78 1405 AAGGUGAUCCAGACUCUGA 196 1405UCUGCUGGAGGCCACAGCA  79 1405 UCUGCUGGAGGCCACAGCA  79 1423UGCUGUGGCCUCCAGCAGA 197 1423 AAACCUCCUCACAGCCCAC  80 1423AAACCUCCUCACAGCCCAC  80 1441 GUGGGCUGUGAGGAGGUUU 198 1441CUGGUCCUCAAGAGGUGCC  81 1441 CUGGUCCUCAAGAGGUGCC  81 1459GGCACCUCUUGAGGACCAG 199 1459 CACGUCUCCACACAUCAGC  82 1459CACGUCUCCACACAUCAGC  82 1477 GCUGAUGUGUGGAGACGUG 200 1477CACAACUACGCAGCGCCUC  83 1477 CACAACUACGCAGCGCCUC  83 1495GAGGCGCUGCGUAGUUGUG 201 1495 CCCUCCACUCGGAAGGACU  84 1495CCCUCCACUCGGAAGGACU  84 1513 AGUCCUUCCGAGUGGAGGG 202 1513UAUCCUGCUGCCAAGAGGG  85 1513 UAUCCUGCUGCCAAGAGGG  85 1531CCCUCUUGGCAGCAGGAUA 203 1531 GUCAAGUUGGACAGUGUCA  86 1531GUCAAGUUGGACAGUGUCA  86 1549 UGACACUGUCCAACUUGAC 204 1549AGAGUCCUGAGACAGAUCA  87 1549 AGAGUCCUGAGACAGAUCA  87 1567UGAUCUGUCUCAGGACUCU 205 1567 AGCAACAACCGAAAAUGCA  88 1567AGCAACAACCGAAAAUGCA  88 1585 UGCAUUUUCGGUUGUUGCU 206 1585ACCAGCCCCAGGUCCUCGG  89 1585 ACCAGCCCCAGGUCCUCGG  89 1603CCGAGGACCUGGGGCUGGU 207 1603 GACACCGAGGAGAAUGUCA  90 1603GACACCGAGGAGAAUGUCA  90 1621 UGACAUUCUCCUCGGUGUC 208 1621AAGAGGCGAACACACAACG  91 1621 AAGAGGCGAACACACAACG  91 1639CGUUGUGUGUUCGCCUCUU 209 1639 GUCUUGGAGCGCCAGAGGA  92 1639GUCUUGGAGCGCCAGAGGA  92 1657 UCCUCUGGCGCUCCAAGAC 210 1657AGGAACGAGCUAAAACGGA  93 1657 AGGAACGAGCUAAAACGGA  93 1675UCCGUUUUAGCUCGUUCCU 211 1675 AGCUUUUUUGCCCUGCGUG  94 1675AGCUUUUUUGCCCUGCGUG  94 1693 CACGCAGGGCAAAAAAGCU 212 1693GACCAGAUCCCGGAGUUGG  95 1693 GACCAGAUCCCGGAGUUGG  95 1711CCAACUCCGGGAUCUGGUC 213 1711 GAAAACAAUGAAAAGGCCC  96 1711GAAAACAAUGAAAAGGCCC  96 1729 GGGCCUUUUCAUUGUUUUC 214 1729CCCAAGGUAGUUAUCCUUA  97 1729 CCCAAGGUAGUUAUCCUUA  97 1747UAAGGAUAACUACCUUGGG 215 1747 AAAAAAGCCACAGCAUACA  98 1747AAAAAAGCCACAGCAUACA  98 1765 UGUAUGCUGUGGCUUUUUU 216 1765AUCCUGUCCGUCCAAGCAG  99 1765 AUCCUGUCCGUCCAAGCAG  99 1783CUGCUUGGACGGACAGGAU 217 1783 GAGGAGCAAAAGCUCAUUU 100 1783GAGGAGCAAAAGCUCAUUU 100 1801 AAAUGAGCUUUUGCUCCUC 218 1801UCUGAAGAGGACUUGUUGC 101 1801 UCUGAAGAGGACUUGUUGC 101 1819GCAACAAGUCCUCUUCAGA 219 1819 CGGAAACGACGAGAACAGU 102 1819CGGAAACGACGAGAACAGU 102 1837 ACUGUUCUCGUCGUUUCCG 220 1837UUGAAACACAAACUUGAAC 103 1837 UUGAAACACAAACUUGAAC 103 1855GUUCAAGUUUGUGUUUCAA 221 1855 CAGCUACGGAACUCUUGUG 104 1855CAGCUACGGAACUCUUGUG 104 1873 CACAAGAGUUCCGUAGCUG 222 1873GCGUAAGGAAAAGUAAGGA 105 1873 GCGUAAGGAAAAGUAAGGA 105 1891UCCUUACUUUUCCUUACGC 223 1891 AAAACGAUUCCUUCUAACA 106 1891AAAACGAUUCCUUCUAACA 106 1909 UGUUAGAAGGAAUCGUUUU 224 1909AGAAAUGUCCUGAGCAAUC 107 1909 AGAAAUGUCCUGAGCAAUC 107 1927GAUUGCUCAGGACAUUUCU 225 1927 CACCUAUGAACUUGUUUCA 108 1927CACCUAUGAACUUGUUUCA 108 1945 UGAAACAAGUUCAUAGGUG 226 1945AAAUGCAUGAUCAAAUGCA 109 1945 AAAUGCAUGAUCAAAUGCA 109 1963UGCAUUUGAUCAUGCAUUU 227 1963 AACCUCACAACCUUGGCUG 110 1963AACCUCACAACCUUGGCUG 110 1981 CAGCCAAGGUUGUGAGGUU 228 1981GAGUCUUGAGACUGAAAGA 111 1981 GAGUCUUGAGACUGAAAGA 111 1999UCUUUCAGUCUCAAGACUC 229 1999 AUUUAGCCAUAAUGUAAAC 112 1999AUUUAGCCAUAAUGUAAAC 112 2017 GUUUACAUUAUGGCUAAAU 230 2017CUGCCUCAAAUUGGACUUU 113 2017 CUGCCUCAAAUUGGACUUU 113 2035AAAGUCCAAUUUGAGGCAG 231 2035 UGGGCAUAAAAGAACUUUU 114 2035UGGGCAUAAAAGAACUUUU 114 2053 AAAAGUUCUUUUAUGCCCA 232 2053UUUAUGCUUACCAUCUUUU 115 2053 UUUAUGCUUACCAUCUUUU 115 2071AAAAGAUGGUAAGCAUAAA 233 2071 UUUUUUUCUUUAACAGAUU 116 2071UUUUUUUCUUUAACAGAUU 116 2089 AAUCUGUUAAAGAAAAAAA 234 2089UUGUAUUUAAGAAUUGUUU 117 2089 UUGUAUUUAAGAAUUGUUU 117 2107AAACAAUUCUUAAAUACAA 235 2103 UGUUUUUAAAAAAUUUUAA 118 2103UGUUUUUAAAAAAUUUUAA 118 2121 UUAAAAUUUUUUAAAAACA 236 NM 005375.1 (hMYB)   3 CGGCAGCGCCCUGCCGACG 237    3 CGGCAGCGCCCUGCCGACG 237   21CGUCGGCAGGGCGCUGCCG 416   21 GCCGGGGAGGGACGCAGGC 238   21GCCGGGGAGGGACGCAGGC 238   39 GCCUGCGUCCCUCCCCGGC 417   39CAGGCGGCGGGCAGCGGGA 239   39 CAGGCGGCGGGCAGCGGGA 239   57UCCCGCUGCCCGCCGCCUG 418   57 AGGCGGCACCCCGGUGCUC 240   57AGGCGGCACCCCGGUGCUC 240   75 GAGCACCGGGGUGCCGCCU 419   75CCCCGCGGCUCUCGGCGGA 241   75 CCCCGCGGCUCUCGGCGGA 241   93UCCGCCGAGAGCCGCGGGG 420   93 AGCCCCGCCGCCCGCCGCG 242   93AGCCCCGCCGCCCGCCGCG 242  111 CGCGGCGGGCGGCGGGGCU 421  111GCCAUGGCCCGAAGACCCC 243  111 GCCAUGGCCCGAAGACCCC 243  129GGGGUCUUCGGGCCAUGGC 422  129 CGGCACAGCAUAUAUAGCA 244  129CGGCACAGCAUAUAUAGCA 244  147 UGCUAUAUAUGCUGUGCCG 423  147AGUGACGAGGAUGAUGAGG 245  147 AGUGACGAGGAUGAUGAGG 245  165CCUCAUCAUCCUCGUCACU 424  165 GACUUUGAGAUGUGUGACC 246  165GACUUUGAGAUGUGUGACC 246  183 GGUCACACAUCUCAAAGUC 425  183CAUGACUAUGAUGGGCUGC 247  183 CAUGACUAUGAUGGGCUGC 247  201GCAGCCCAUCAUAGUCAUG 426  201 CUUCCCAAGUCUGGAAAGC 248  201CUUCCCAAGUCUGGAAAGC 248  219 GCUUUCCAGACUUGGGAAG 427  219CGUCACUUGGGGAAAACAA 249  219 CGUCACUUGGGGAAAACAA 249  237UUGUUUUCCCCAAGUGACG 428  237 AGGUGGACCCGGGAAGAGG 250  37AGGUGGACCCGGGAAGAGG 250  255 CCUCUUCCCGGGUCCACCU 429  255GAUGAAAAACUGAAGAAGC 251  255 GAUGAAAAACUGAAGAAGC 251  273GCUUCUUCAGUUUUUCAUC 430  273 CUGGUGGAACAGAAUGGAA 252  273CUGGUGGAACAGAAUGGAA 252  291 UUCCAUUCUGUUCCACCAG 431  291ACAGAUGACUGGAAAGUUA 253  291 ACAGAUGACUGGAAAGUUA 253  309UAACUUUCCAGUCAUCUGU 432  309 AUUGCCAAUUAUCUCCCGA 254  309AUUGCCAAUUAUCUCCCGA 254  327 UCGGGAGAUAAUUGGCAAU 433  327AAUCGAACAGAUGUGCAGU 255  327 AAUCGAACAGAUGUGCAGU 255  345ACUGCACAUCUGUUCGAUU 434  345 UGCCAGCACCGAUGGCAGA 256  345UGCCAGCACCGAUGGCAGA 256  363 UCUGCCAUCGGUGCUGGCA 435  363AAAGUACUAAACCCUGAGC 257  363 AAAGUACUAAACCCUGAGC 257  381GCUCAGGGUUUAGUACUUU 436  381 CUCAUCAAGGGUCCUUGGA 258  381CUCAUCAAGGGUCCUUGGA 258  399 UCCAAGGACCCUUGAUGAG 437  399ACCAAAGAAGAAGAUCAGA 259  399 ACCAAAGAAGAAGAUCAGA 259  417UCUGAUCUUCUUCUUUGGU 438  417 AGAGUGAUAGAGCUUGUAC 260  417AGAGUGAUAGAGCUUGUAC 260  435 GUACAAGCUCUAUCACUCU 439  435CAGAAAUACGGUCCGAAAC 261  435 CAGAAAUACGGUCCGAAAC 261  453GUUUCGGACCGUAUUUCUG 440  453 CGUUGGUCUGUUAUUGCCA 262  453CGUUGGUCUGUUAUUGCCA 262  471 UGGCAAUAACAGACCAACG 441  471AAGCACUUAAAGGGGAGAA 263  471 AAGCACUUAAAGGGGAGAA 263  489UUCUCCCCUUUAAGUGCUU 442  489 AUUGGAAAACAAUGUAGGG 264  489AUUGGAAAACAAUGUAGGG 264  507 CCCUACAUUGUUUUCCAAU 443  507GAGAGGUGGCAUAACCACU 265  507 GAGAGGUGGCAUAACCACU 265  525AGUGGUUAUGCCACCUCUC 444  525 UUGAAUCCAGAAGUUAAGA 266  525UUGAAUCCAGAAGUUAAGA 266  543 UCUUAACUUCUGGAUUCAA 445  543AAAACCUCCUGGACAGAAG 267  543 AAAACCUCCUGGACAGAAG 267  561CUUCUGUCCAGGAGGUUUU 446  561 GAGGAAGACAGAAUUAUUU 268  561GAGGAAGACAGAAUUAUUU 268  579 AAAUAAUUCUGUCUUCCUC 447  579UACCAGGCACACAAGAGAC 269  579 UACCAGGCACACAAGAGAC 269  597GUCUCUUGUGUGCCUGGUA 448  597 CUGGGGAACAGAUGGGCAG 270  597CUGGGGAACAGAUGGGCAG 270  615 CUGCCCAUCUGUUCCCCAG 449  615GAAAUCGCAAAGCUACUGC 271  615 GAAAUCGCAAAGCUACUGC 271  633GCAGUAGCUUUGCGAUUUC 450  633 CCUGGACGAACUGAUAAUG 272  633CCUGGACGAACUGAUAAUG 272  651 CAUUAUCAGUUCGUCCAGG 451  651GCUAUCAAGAACCACUGGA 273  651 GCUAUCAAGAACCACUGGA 273  669UCCAGUGGUUCUUGAUAGC 452  669 AAUUCUACAAUGCGUCGGA 274  669AAUUCUACAAUGCGUCGGA 274  687 UCCGACGCAUUGUAGAAUU 453  687AAGGUCGAACAGGAAGGUU 275  687 AAGGUCGAACAGGAAGGUU 275  705AACCUUCCUGUUCGACCUU 454  705 UAUCUGCAGGAGUCUUCAA 276  705UAUCUGCAGGAGUCUUCAA 276  723 UUGAAGACUCCUGCAGAUA 455  723AAAGCCAGCCAGCCAGCAG 277  723 AAAGCCAGCCAGCCAGCAG 277  741CUGCUGGCUGGCUGGCUUU 456  741 GUGGCCACAAGCUUCCAGA 278  741GUGGCCACAAGCUUCCAGA 278  759 UCUGGAAGCUUGUGGCCAC 457  759AAGAACAGUCAUUUGAUGG 279  759 AAGAACAGUCAUUUGAUGG 279  777CCAUCAAAUGACUGUUCUU 458  777 GGUUUUGCUCAGGCUCCGC 280  777GGUUUUGCUCAGGCUCCGC 280  795 GCGGAGCCUGAGCAAAACC 459  795CCUACAGCUCAACUCCCUG 281  795 CCUACAGCUCAACUCCCUG 281  813CAGGGAGUUGAGCUGUAGG 460  813 GCCACUGGCCAGCCCACUG 282  813GCCACUGGCCAGCCCACUG 282  831 CAGUGGGCUGGCCAGUGGC 461  831GUUAACAACGACUAUUCCU 283  831 GUUAACAACGACUAUUCCU 283  849AGGAAUAGUCGUUGUUAAC 462  849 UAUUACCACAUUUCUGAAG 284  849UAUUACCACAUUUCUGAAG 284  867 CUUCAGAAAUGUGGUAAUA 463  867GCACAAAAUGUCUCCAGUC 285  867 GCACAAAAUGUCUCCAGUC 285  885GACUGGAGACAUUUUGUGC 464  885 CAUGUUCCAUACCCUGUAG 286  885CAUGUUCCAUACCCUGUAG 286  903 CUACAGGGUAUGGAACAUG 465  903GCGUUACAUGUAAAUAUAG 287  903 GCGUUACAUGUAAAUAUAG 287  921CUAUAUUUACAUGUAACGC 466  921 GUCAAUGUCCCUCAGCCAG 288  921GUCAAUGUCCCUCAGCCAG 288  939 CUGGCUGAGGGACAUUGAC 467  939GCUGCCGCAGCCAUUCAGA 289  939 GCUGCCGCAGCCAUUCAGA 289  957UCUGAAUGGCUGCGGCAGC 468  957 AGACACUAUAAUGAUGAAG 290  957AGACACUAUAAUGAUGAAG 290  975 CUUCAUCAUUAUAGUGUCU 469  975GACCCUGAGAAGGAAAAGC 291  975 GACCCUGAGAAGGAAAAGC 291  993GCUUUUCCUUCUCAGGGUC 470  993 CGAAUAAAGGAAUUAGAAU 292  993CGAAUAAAGGAAUUAGAAU 292 1011 AUUCUAAUUCCUUUAUUCG 471 1011UUGCUCCUAAUGUCAACCG 293 1011 UUGCUCCUAAUGUCAACCG 293 1029CGGUUGACAUUAGGAGCAA 472 1029 GAGAAUGAGCUAAAAGGAC 294 1029GAGAAUGAGCUAAAAGGAC 294 1047 GUCCUUUUAGCUCAUUCUC 473 1047CAGCAGGUGCUACCAACAC 295 1047 CAGCAGGUGCUACCAACAC 295 1065GUGUUGGUAGCACCUGCUG 474 1065 CAGAACCACACAUGCAGCU 296 1065CAGAACCACACAUGCAGCU 296 1083 AGCUGCAUGUGUGGUUCUG 475 1083UACCCCGGGUGGCACAGCA 297 1083 UACCCCGGGUGGCACAGCA 297 1101UGCUGUGCCACCCGGGGUA 476 1101 ACCACCAUUGCCGACCACA 298 1101ACCACCAUUGCCGACCACA 298 1119 UGUGGUCGGCAAUGGUGGU 477 1119ACCAGACCUCAUGGAGACA 299 1119 ACCAGACCUCAUGGAGACA 299 1137UGUCUCCAUGAGGUCUGGU 478 1137 AGUGCACCUGUUUCCUGUU 300 1137AGUGCACCUGUUUCCUGUU 300 1155 AACAGGAAACAGGUGCACU 479 1155UUGGGAGAACACCACUCCA 301 1155 UUGGGAGAACACCACUCCA 301 1173UGGAGUGGUGUUCUCCCAA 480 1173 ACUCCAUCUCUGCCAGCGG 302 1173ACUCCAUCUCUGCCAGCGG 302 1191 CCGCUGGCAGAGAUGGAGU 481 1191GAUCCUGGCUCCCUACCUG 303 1191 GAUCCUGGCUCCCUACCUG 303 1209CAGGUAGGGAGCCAGGAUC 482 1209 GAAGAAAGCGCCUCGCCAG 304 1209GAAGAAAGCGCCUCGCCAG 304 1227 CUGGCGAGGCGCUUUCUUC 483 1227GCAAGGUGCAUGAUCGUCC 305 1227 GCAAGGUGCAUGAUCGUCC 305 1245GGACGAUCAUGCACCUUGC 484 1245 CACCAGGGCACCAUUCUGG 306 1245CACCAGGGCACCAUUCUGG 306 1263 CCAGAAUGGUGCCCUGGUG 485 1263GAUAAUGUUAAGAACCUCU 307 1263 GAUAAUGUUAAGAACCUCU 307 1281AGAGGUUCUUAACAUUAUC 486 1281 UUAGAAUUUGCAGAAACAC 308 1281UUAGAAUUUGCAGAAACAC 308 1299 GUGUUUCUGCAAAUUCUAA 487 1299CUCCAAUUUAUAGAUUCUU 309 1299 CUCCAAUUUAUAGAUUCUU 309 1317AAGAAUCUAUAAAUUGGAG 488 1317 UUCUUAAACACUUCCAGUA 310 1317UUCUUAAACACUUCCAGUA 310 1335 UACUGGAAGUGUUUAAGAA 489 1335AACCAUGAAAACUCAGACU 311 1335 AACCAUGAAAACUCAGACU 311 1353AGUCUGAGUUUUCAUGGUU 490 1353 UUGGAAAUGCCUUCUUUAA 312 1353UUGGAAAUGCCUUCUUUAA 312 1371 UUAAAGAAGGCAUUUCCAA 491 1371ACUUCCACCCCCCUCAUUG 313 1371 ACUUCCACCCCCCUCAUUG 313 1389CAAUGAGGGGGGUGGAAGU 492 1389 GGUCACAAAUUGACUGUUA 314 1389GGUCACAAAUUGACUGUUA 314 1407 UAACAGUCAAUUUGUGACC 493 1407ACAACACCAUUUCAUAGAG 315 1407 ACAACACCAUUUCAUAGAG 315 1425CUCUAUGAAAUGGUGUUGU 494 1425 GACCAGACUGUGAAAACUC 316 1425GACCAGACUGUGAAAACUC 316 1443 GAGUUUUCACAGUCUGGUC 495 1443CAAAAGGAAAAUACUGUUU 317 1443 CAAAAGGAAAAUACUGUUU 317 1461AAACAGUAUUUUCCUUUUG 496 1461 UUUAGAACCCCAGCUAUCA 318 1461UUUAGAACCCCAGCUAUCA 318 1479 UGAUAGCUGGGGUUCUAAA 497 1479AAAAGGUCAAUCUUAGAAA 319 1479 AAAAGGUCAAUCUUAGAAA 319 1497UUUCUAAGAUUGACCUUUU 498 1497 AGCUCUCCAAGAACUCCUA 320 1497AGCUCUCCAAGAACUCCUA 320 1515 UAGGAGUUCUUGGAGAGCU 499 1515ACACCAUUCAAACAUGCAC 321 1515 ACACCAUUCAAACAUGCAC 321 1533GUGCAUGUUUGAAUGGUGU 500 1533 CUUGCAGCUCAAGAAAUUA 322 1533CUUGCAGCUCAAGAAAUUA 322 1551 UAAUUUCUUGAGCUGCAAG 501 1551AAAUACGGUCCCCUGAAGA 323 1551 AAAUACGGUCCCCUGAAGA 323 1569UCUUCAGGGGACCGUAUUU 502 1569 AUGCUACCUCAGACACCCU 324 1569AUGCUACCUCAGACACCCU 324 1587 AGGGUGUCUGAGGUAGCAU 503 1587UCUCAUCUAGUAGAAGAUC 325 1587 UCUCAUCUAGUAGAAGAUC 325 1605GAUCUUCUACUAGAUGAGA 504 1605 CUGCAGGAUGUGAUCAAAC 326 1605CUGCAGGAUGUGAUCAAAC 326 1623 GUUUGAUCACAUCCUGCAG 505 1623CAGGAAUCUGAUGAAUCUG 327 1623 CAGGAAUCUGAUGAAUCUG 327 1641CAGAUUCAUCAGAUUCCUG 506 1641 GGAUUUGUUGCUGAGUUUC 328 1641GGAUUUGUUGCUGAGUUUC 328 1659 GAAACUCAGCAACAAAUCC 507 1659CAAGAAAAUGGACCACCCU 329 1659 CAAGAAAAUGGACCACCCU 329 1677AGGGUGGUCCAUUUUCUUG 508 1677 UUACUGAAGAAAAUCAAAC 330 1677UUACUGAAGAAAAUCAAAC 330 1695 GUUUGAUUUUCUUCAGUAA 509 1695CAAGAGGUGGAAUCUCCAA 331 1695 CAAGAGGUGGAAUCUCCAA 331 1713UUGGAGAUUCCACCUCUUG 510 1713 ACUGAUAAAUCAGGAAACU 332 1713ACUGAUAAAUCAGGAAACU 332 1731 AGUUUCCUGAUUUAUCAGU 511 1731UUCUUCUGCUCACACCACU 333 1731 UUCUUCUGCUCACACCACU 333 1749AGUGGUGUGAGCAGAAGAA 512 1749 UGGGAAGGGGACAGUCUGA 334 1749UGGGAAGGGGACAGUCUGA 334 1767 UCAGACUGUCCCCUUCCCA 513 1767AAUACCCAACUGUUCACGC 335 1767 AAUACCCAACUGUUCACGC 335 1785GCGUGAACAGUUGGGUAUU 514 1785 CAGACCUCGCCUGUGCGAG 336 1785CAGACCUCGCCUGUGCGAG 336 1803 CUCGCACAGGCGAGGUCUG 515 1803GAUGCACCGAAUAUUCUUA 337 1803 GAUGCACCGAAUAUUCUUA 337 1821UAAGAAUAUUCGGUGCAUC 516 1821 ACAAGCUCCGUUUUAAUGG 338 1821ACAAGCUCCGUUUUAAUGG 338 1839 CCAUUAAAACGGAGCUUGU 517 1839GCACCAGCAUCAGAAGAUG 339 1839 GCACCAGCAUCAGAAGAUG 339 1857CAUCUUCUGAUGCUGGUGC 518 1857 GAAGACAAUGUUCUCAAAG 340 1857GAAGACAAUGUUCUCAAAG 340 1875 CUUUGAGAACAUUGUCUUC 519 1875GCAUUUACAGUACCUAAAA 341 1875 GCAUUUACAGUACCUAAAA 341 1893UUUUAGGUACUGUAAAUGC 520 1893 AACAGGUCCCUGGCGAGCC 342 1893AACAGGUCCCUGGCGAGCC 342 1911 GGCUCGCCAGGGACCUGUU 521 1911CCCUUGCAGCCUUGUAGCA 343 1911 CCCUUGCAGCCUUGUAGCA 343 1929UGCUACAAGGCUGCAAGGG 522 1929 AGUACCUGGGAACCUGCAU 344 1929AGUACCUGGGAACCUGCAU 344 1947 AUGCAGGUUCCCAGGUACU 523 1947UCCUGUGGAAAGAUGGAGG 345 1947 UCCUGUGGAAAGAUGGAGG 345 1965CCUCCAUCUUUCCACAGGA 524 1965 GAGCAGAUGACAUCUUCCA 346 1965GAGCAGAUGACAUCUUCCA 346 1983 UGGAAGAUGUCAUCUGCUC 525 1983AGUCAAGCUCGUAAAUACG 347 1983 AGUCAAGCUCGUAAAUACG 347 2001CGUAUUUACGAGCUUGACU 526 2001 GUGAAUGCAUUCUCAGCCC 348 2001GUGAAUGCAUUCUCAGCCC 348 2019 GGGCUGAGAAUGCAUUCAC 527 2019CGGACGCUGGUCAUGUGAG 349 2019 CGGACGCUGGUCAUGUGAG 349 2037CUCACAUGACCAGCGUCCG 528 2037 GACAUUUCCAGAAAAGCAU 350 2037GACAUUUCCAGAAAAGCAU 350 2055 AUGCUUUUCUGGAAAUGUC 529 2055UUAUGGUUUUCAGAACAGU 351 2055 UUAUGGUUUUCAGAACAGU 351 2073ACUGUUCUGAAAACCAUAA 530 2073 UUCAAGUUGACUUGGGAUA 352 2073UUCAAGUUGACUUGGGAUA 352 2091 UAUCCCAAGUCAACUUGAA 531 2091AUAUCAUUCCUCAACAUGA 353 2091 AUAUCAUUCCUCAACAUGA 353 2109UCAUGUUGAGGAAUGAUAU 532 2109 AAACUUUUCAUGAAUGGGA 354 2109AAACUUUUCAUGAAUGGGA 354 2127 UCCCAUUCAUGAAAAGUUU 533 2127AGAAGAACCUAUUUUUGUU 355 2127 AGAAGAACCUAUUUUUGUU 355 2145AACAAAAAUAGGUUCUUCU 534 2145 UGUGGUACAACAGUUGAGA 356 2145UGUGGUACAACAGUUGAGA 356 2163 UCUCAACUGUUGUACCACA 535 2163AGCACGACCAAGUGCAUUU 357 2163 AGCACGACCAAGUGCAUUU 357 2181AAAUGCACUUGGUCGUGCU 536 2181 UAGUUGAAUGAAGUCUUCU 358 2181UAGUUGAAUGAAGUCUUCU 358 2199 AGAAGACUUCAUUCAACUA 537 2199UUGGAUUUCACCCAACUAA 359 2199 UUGGAUUUCACCCAACUAA 359 2217UUAGUUGGGUGAAAUCCAA 538 2217 AAAGGAUUUUUAAAAAUAA 360 2217AAAGGAUUUUUAAAAAUAA 360 2235 UUAUUUUUAAAAAUCCUUU 539 2235AAUAACAGUCUUACCUAAA 361 2235 AAUAACAGUCUUACCUAAA 361 2253UUUAGGUAAGACUGUUAUU 540 2253 AUUAUUAGGUAAUGAAUUG 362 2253AUUAUUAGGUAAUGAAUUG 362 2271 CAAUUCAUUACCUAAUAAU 541 2271GUAGCCAGUUGUUAAUAUC 363 2271 GUAGCCAGUUGUUAAUAUC 363 2289GAUAUUAACAACUGGCUAC 542 2289 CUUAAUGCAGAUUUUUUUA 364 2289CUUAAUGCAGAUUUUUUUA 364 2307 UAAAAAAAUCUGCAUUAAG 543 2307AAAAAAAAACAUAAAAUGA 365 2307 AAAAAAAAACAUAAAAUGA 365 2325UCAUUUUAUGUUUUUUUUU 544 2325 AUUUAUCUGGUAUUUUAAA 366 2325AUUUAUCUGGUAUUUUAAA 366 2343 UUUAAAAUACCAGAUAAAU 545 2343AGGAUCCAACAGAUCAGUA 367 2343 AGGAUCCAACAGAUCAGUA 367 2361UACUGAUCUGUUGGAUCCU 546 2361 AUUUUUUCCUGUGAUGGGU 368 2361AUUUUUUCCUGUGAUGGGU 368 2379 ACCCAUCACAGGAAAAAAU 547 2379UUUUUUGAAAUUUGACACA 369 2379 UUUUUUGAAAUUUGACACA 369 2397UGUGUCAAAUUUCAAAAAA 548 2397 AUUAAAAGGUACUCCAGUA 370 2397AUUAAAAGGUACUCCAGUA 370 2415 UACUGGAGUACCUUUUAAU 549 2415AUUUCACUUUUCUCGAUCA 371 2415 AUUUCACUUUUCUCGAUCA 371 2433UGAUCGAGAAAAGUGAAAU 550 2433 ACUAAACAUAUGCAUAUAU 372 2433ACUAAACAUAUGCAUAUAU 372 2451 AUAUAUGCAUAUGUUUAGU 551 2451UUUUUAAAAAUCAGUAAAA 373 2451 UUUUUAAAAAUCAGUAAAA 373 2469UUUUACUGAUUUUUAAAAA 552 2469 AGCAUUACUCUAAGUGUAG 374 2469AGCAUUACUCUAAGUGUAG 374 2487 CUACACUUAGAGUAAUGCU 553 2487GACUUAAUACCAUGUGACA 375 2487 GACUUAAUACCAUGUGACA 375 2505UGUCACAUGGUAUUAAGUC 554 2505 AUUUAAUCCAGAUUGUAAA 376 2505AUUUAAUCCAGAUUGUAAA 376 2523 UUUACAAUCUGGAUUAAAU 555 2523AUGCUCAUUUAUGGUUAAU 377 2523 AUGCUCAUUUAUGGUUAAU 377 2541AUUAACCAUAAAUGAGCAU 556 2541 UGACAUUGAAGGUACAUUU 378 2541UGACAUUGAAGGUACAUUU 378 2559 AAAUGUACCUUCAAUGUCA 557 2559UAUUGUACCAAACCAUUUU 379 2559 UAUUGUACCAAACCAUUUU 379 2577AAAAUGGUUUGGUACAAUA 558 2577 UAUGAGUUUUCUGUUAGCU 380 2577UAUGAGUUUUCUGUUAGCU 380 2595 AGCUAACAGAAAACUCAUA 559 2595UUGCUUUAAAAAUUAUUAC 381 2595 UUGCUUUAAAAAUUAUUAC 381 2613GUAAUAAUUUUUAAAGCAA 560 2613 CUGUAAGAAAUAGUUUUAU 382 2613CUGUAAGAAAUAGUUUUAU 382 2631 AUAAAACUAUUUCUUACAG 561 2631UAAAAAAUUAUAUUUUUAU 383 2631 UAAAAAAUUAUAUUUUUAU 383 2649AUAAAAAUAUAAUUUUUUA 562 2649 UUCAGUAAUUUAAUUUUGU 384 2649UUCAGUAAUUUAAUUUUGU 384 2667 ACAAAAUUAAAUUACUGAA 563 2667UAAAUGCCAAAUGAAAAAC 385 2667 UAAAUGCCAAAUGAAAAAC 385 2685GUUUUUCAUUUGGCAUUUA 564 2685 CGUUUUUUGCUGCUAUGGU 386 2685CGUUUUUUGCUGCUAUGGU 386 2703 ACCAUAGCAGCAAAAAACG 565 2703UCUUAGCCUGUAGACAUGC 387 2703 UCUUAGCCUGUAGACAUGC 387 2721GCAUGUCUACAGGCUAAGA 566 2721 CUGCUAGUAUCAGAGGGGC 388 2721CUGCUAGUAUCAGAGGGGC 388 2739 GCCCCUCUGAUACUAGCAG 567 2739CAGUAGAGCUUGGACAGAA 389 2739 CAGUAGAGCUUGGACAGAA 389 2757UUCUGUCCAAGCUCUACUG 568 2757 AAGAAAAGAAACUUGGUGU 390 2757AAGAAAAGAAACUUGGUGU 390 2775 ACACCAAGUUUCUUUUCUU 569 2775UUAGGUAAUUGACUAUGCA 391 2775 UUAGGUAAUUGACUAUGCA 391 2793UGCAUAGUCAAUUACCUAA 570 2793 ACUAGUAUUUCAGACUUUU 392 2793ACUAGUAUUUCAGACUUUU 392 2811 AAAAGUCUGAAAUACUAGU 571 2811UUAAUUUUAUAUAUAUAUA 393 2811 UUAAUUUUAUAUAUAUAUA 393 2829UAUAUAUAUAUAAAAUUAA 572 2829 ACAUUUUUUUUCCUUCUGC 394 2829ACAUUUUUUUUCCUUCUGC 394 2847 GCAGAAGGAAAAAAAAUGU 573 2847CAAUACAUUUGAAAACUUG 395 2847 CAAUACAUUUGAAAACUUG 395 2865CAAGUUUUCAAAUGUAUUG 574 2865 GUUUGGGAGACUCUGCAUU 396 2865GUUUGGGAGACUCUGCAUU 396 2883 AAUGCAGAGUCUCCCAAAC 575 2883UUUUUAUUGUGGUUUUUUU 397 2883 UUUUUAUUGUGGUUUUUUU 397 2901AAAAAAACCACAAUAAAAA 576 2901 UGUUAUUGUUGGUUUAUAC 398 2901UGUUAUUGUUGGUUUAUAC 398 2919 GUAUAAACCAACAAUAACA 577 2919CAAGCAUGCGUUGCACUUC 399 2919 CAAGCAUGCGUUGCACUUC 399 2937GAAGUGCAACGCAUGCUUG 578 2937 CUUUUUUGGGAGAUGUGUG 400 2937CUUUUUUGGGAGAUGUGUG 400 2955 CACACAUCUCCCAAAAAAG 579 2955GUUGUUCAUGUUCUAUGUU 401 2955 GUUGUUCAUGUUCUAUGUU 401 2973AACAUAGAACAUGAACAAC 580 2973 UUUGUUUUGUGUGUAGCCU 402 2973UUUGUUUUGUGUGUAGCCU 402 2991 AGGCUACACACAAAACAAA 581 2991UGACUGUUUUAUAAUUUGG 403 2991 UGACUGUUUUAUAAUUUGG 403 3009CCAAAUUAUAAAACAGUCA 582 3009 GGAGUUCUCGAUUUGAUCC 404 3009GGAGUUCUCGAUUUGAUCC 404 3027 GGAUCAAAUCGAGAACUCC 583 3027CGCAUCCCCUGUGGUUUCU 405 3027 CGCAUCCCCUGUGGUUUCU 405 3045AGAAACCACAGGGGAUGCG 584 3045 UAAGUGUAUGGUCUCAGAA 406 3045UAAGUGUAUGGUCUCAGAA 406 3063 UUCUGAGACCAUACACUUA 585 3063ACUGUUGCAUGGAUCCUGU 407 3063 ACUGUUGCAUGGAUCCUGU 407 3081ACAGGAUCCAUGCAACAGU 586 3081 UGUUUGCAACUGGGGAGAC 408 3081UGUUUGCAACUGGGGAGAC 408 3099 GUCUCCCCAGUUGCAAACA 587 3099CAGAAACUGUGGUUGAUAG 409 3099 CAGAAACUGUGGUUGAUAG 409 3117CUAUCAACCACAGUUUCUG 588 3117 GCCAGUCACUGCCUUAAGA 410 3117GCCAGUCACUGCCUUAAGA 410 3135 UCUUAAGGCAGUGACUGGC 589 3135AACAUUUGAUGCAAGAUGG 411 3135 AACAUUUGAUGCAAGAUGG 411 3153CCAUCUUGCAUCAAAUGUU 590 3153 GCCAGCACUGAACUUUUGA 412 3153GCCAGCACUGAACUUUUGA 412 3171 UCAAAAGUUCAGUGCUGGC 591 3171AGAUAUGACGGUGUACUUA 413 3171 AGAUAUGACGGUGUACUUA 413 3189UAAGUACACCGUCAUAUCU 592 3189 ACUGCCUUGUAGCAAAAUA 414 3189ACUGCCUUGUAGCAAAAUA 414 3207 UAUUUUGCUACAAGGCAGU 593 3205AUAAAGAUGUGCCCUUAUU 415 3205 AUAAAGAUGUGCCCUUAUU 415 3223AAUAAGGGCACAUCUUUAU 594 The 3′-ends of the Upper sequence and the Lowersequence of the siNA construct can include an overhang sequence, forexample about 1, 2, 3, or 4 nucleotides in length, preferably 2nucleotides in length, wherein the overhanging sequence of the lowersequence is optionally complementary to a portion of the targetsequence. The upper sequence is also referred to as the sense strand,whereas the lower sequence is also referred to as the antisense strand.The upper and lowersequences in the Table can further comprise achemical modification having Formulae I-VII of any combination thereof.

TABLE III Myc and Myb Synthetic Modified siNA constructs Target PosTarget Seq ID Aliases Sequence Seq ID Myc Synthetic Modified siNAconstructs 1524 CAAGAGGGUCAAGUUGGACAGUG 595 MYC: 1526U21 siNA senseAGAGGGUCAAGUUGGACAGTT 606 1778 AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1780U21siNA sense GCAGAGGAGCAAAAGCUCATT 607 1859 UACGGAACUCUUGUGCGUAAGGA 597MYC: 1861U21 siNA sense CGGAACUCUUGUGCGUAAGTT 608 1969ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1971U21 siNA senseAACCUUGGCUGAGUCUUGATT 609 1524 CAAGAGGGUCAAGUUGGACAGUG 595 MYC: 1544L21siNA (1526C) CUGUCCAACUUGACCCUCUTT 610 antisense 1778AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1798L21 siNA (1780C)UGAGCUUUUGCUCCUCUGCTT 611 antisense 1859 UACGGAACUCUUGUGCGUAAGGA 597MYC: 1879L21 siNA (1861C) CUUACGCACAAGAGUUCCGTT 612 antisense 1969ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1989L21 siNA (1971C)UCAAGACUCAGCCAAGGUUTT 613 antisense 1524 CAAGAGGGUCAAGUUGGACAGUG 595MYC: 1526U21 siNA stab04 B AGAGGGucAAGuuGGAcAGTT B 614 sense 1778AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1780U21 siNA stab04 BGcAGAGGAGcAAAAGcucATT B 615 sense 1859 UACGGAACUCUUGUGCGUAAGGA 597 MYC:1861U21 siNA stab04 B cGGAAcucuuGuGcGuAAGTT B 616 sense 1969ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1971U21 siNA stab04 BAAccuuGGcuGAGucuuGATTB 617 sense 1524 CAAGAGGGUCAAGUUGGACAGUG 595 MYC:1544L21 siNA (1526C) cuGuccAAcuuGAcccucuTsT 618 stab05 antisense 1778AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1798L21 siNA (1780C)uGAGcuuuuGcuccucuGcTsT 619 stab05 antisense 1859 UACGGAACUCUUGUGCGUAAGGA597 MYC: 1879L21 siNA (1861C) cuuAcGcAcAAGAGuuccGTsT 620 stab05antisense 1969 ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1989L21 siNA (1971C)ucAAGAcucAGccAAGGuuTsT 621 stab05 antisense 1524 CAAGAGGGUCAAGUUGGACAGUG595 MYC: 1526U21 siNA stab07 B AGAGGGucAAGuuGGAcAGTT B 622 sense 1778AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1780U21 siNA stab07 BGcAGAGGAGcAAAAGcucATT B 623 sense 1859 UACGGAACUCUUGUGCGUAAGGA 597 MYC:1861U21 siNA stab07 B cGGAAcucuuGuGcGuAAGTT B 624 sense 1969ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1971U21 siNA stab07 BAAccuuGGcuGAGucuuGATT B 625 sense 1524 CAAGAGGGUCAAGUUGGACAGUG 595 MYC:1544L21 siNA (1526C) cuGuccAAcuuGAcccucuTsT 626 stab11 antisense 1778AAGCAGAGGAGCAAAAGCUCAUU 596 MYC: 1798L21 siNA (1780C)uGAGcuuuuGcuccucuGcTsT 627 stab11 antisense 1859 UACGGAACUCUUGUGCGUAAGGA597 MYC: 1879L21 siNA (1861C) cuuAcGcAcAAGAGuuccGTsT 628 stab11antisense 1969 ACAACCUUGGCUGAGUCUUGAGA 598 MYC: 1989L21 siNA (1971C)ucAAGAcucAGccAAGGuuTsT 629 stab11 antisense Myc Synthetic Modified siNAconstructs Target Seq ID Cmpd # Alias Sequence Seq IDCAAGAGGGUCAAGUUGGACAGUG 595 30993 MYC: 1526U21 siNA senseAGAGGGUCAAGUUGGACAGTT 606 CAAGAGGGUCAAGUUGGACAGUG 595 30825 MYC: 1526U21siNA stab04 sense B AGAGGGucAAGuuGGAcAGTT B 614 AAGCAGAGGAGCAAAAGCUCAUU596 30826 MYC: 178CU21 siNA stab04 sense B GcAGAGGAGcAAAAGcucATT B 615UACGGAACUCUUGUGCGUAAGGA 597 30827 MYC: 1861U21 siNA stab04 sense BcGGAAcucuuGuGcGuAAGTT B 616 ACAACCUUGGCUGAGUCUUGAGA 598 30828 MYC:1971U21 siNA stab04 sense B AAccuuGGcuGAGucuuGATT B 617CAAGAGGGUCAAGUUGGACAGUG 595 30829 MYC: 1544L21 siNA (1526C) stab05cuGuccAAcuuGAcccucuTsT 618 antisense AAGCAGAGGAGCAAAAGCUCAUU 596 30830MYC: 1798L21 siNA (1780C) stab05 uGAGcuuuuGcuccucuGcTsT 619 antisenseUACGGAACUCUUGUGCGUAAGGA 597 30831 MYC: 1879L21 siNA (1861C) stab05cuuAcGcAcAAGAGuuccGTsT 620 antisense ACAACCUUGGCUGAGUCUUGAGA 598 30832MYC: 1989L21 siNA (1971C) stab05 ucAAGAcucAGccAAGGuuTsT 621 antisenseAAGCAGAGGAGCAAAAGCUCAUU 596 30994 MYC: 178CU21 siNA senseGCAGAGGAGCAAAAGCUCATT 607 UACGGAACUCUUGUGCGUAAGGA 597 30995 MYC: 1861U21siNA sense CGGAACUCUUGUGCGUAAGTT 608 ACAACCUUGGCUGAGUCUUGAGA 598 30996MYC: 1971U21 siNA sense AACCUUGGCUGAGUCUUGATT 609CAAGAGGGUCAAGUUGGACAGUG 595 31069 MYC: 1544L21 siNA (1526C)CUGUCCAACUUGACCCUCUTT 610 antisense AAGCAGAGGAGCAAAAGCUCAUU 596 31070MYC: 1798L21 siNA (1780C) UGAGCUUUUGCUCCUCUGCTT 611 antisenseUACGGAACUCUUGUGCGUAAGGA 597 31071 MYC: 1879L21 siNA (1861C)CUUACGCACAAGAGUUCCGTT 612 antisense ACAACCUUGGCUGAGUCUUGAGA 598 31072MYC: 1989L21 siNA (1971C) UCAAGACUCAGCCAAGGUUTT 613 antisenseACAACCUUGGCUGAGUCUUGAGA 598 31377 MYC: 1971U21 siNA stab04 sense BAAccuuGGcuGAGucuuGATT B 617 ACAACCUUGGCUGAGUCUUGAGA 598 31380 MYC:1989L21 siNA (1971C) stab05 ucAAGAcucAGccAAGGuuTsT 621 antisenseACAACCUUGGCUGAGUCUUGAGA 598 31383 MYC: 1971U21 siNA stab07 sense BAAccuuGGcuGAGucuuGATTB 625 ACAACCUUGGCUGAGUCUUGAGA 598 31386 MYC:1989L21 siNA (1971C) stab11 ucAAGAcucAGccAAGGuuTsT 629 antisenseACAACCUUGGCUGAGUCUUGAGA 598 31389 MYC: 1971U21 siNA inv stab04 BAGuucuGAGucGGuuccAATT B 652 sense ACAACCUUGGCUGAGUCUUGAGA 598 31392 MYC:1989L21 siNA (1971C) inv uuGGAAccGAcucAGAAcuTsT 653 stab05 antisenseACAACCUUGGCUGAGUCUUGAGA 598 31395 MYC: 1971U21 siNA inv stab07 BAGuucuGAGucGGuuccAATT B 654 sense ACAACCUUGGCUGAGUCUUGAGA 598 31398 MYC:1989L21 siNA (1971C) inv uuGGAAccGAcucAGAAcuTsT 655 stab11 antisense MybSynthetic Modified siNA constructs Target Pos Target Seq ID AliasesSequence Seq ID 146 CAGUGACGAGGAUGAUGAGGACU 599 MYB: 148U21 siNA senseGUGACGAGGAUGAUGAGGATT 630 455 UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 457U21siNA sense GGUCUGUUAUUGCCAAGCATT 631 706 AUCUGCAGGAGUCUUCAAAAGCC 601MYB: 708U21 siNA sense CUGCAGGAGUCUUCAAAAGTT 632 1051 AGGUGCUACCAACACAGAACCAC 602 MYB: 1053U21 siNA senseGUGCUACCAACACAGAACCTT 633 146 CAGUGACGAGGAUGAUGAGGACU 599 MYB: 166L21siNA (148C) UCCUCAUCAUCCUCGUCACTT 603 antisense 455UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 475L21 siNA (457C)UGCUUGGCAAUAACAGACCTT 604 antisense 706 AUCUGCAGGAGUCUUCAAAAGCC 601 MYB:726L21 siNA (708C) CUUUUGAAGACUCCUGCAGTT 634 antisense 1051 AGGUGCUACCAACACAGAACCAC 602 MYB: 1071L21 siNA (1053C)GGUUCUGUGUUGGUAGCACTT 635 antisense 146 CAGUGACGAGGAUGAUGAGGACU 599 MYB:148U21 siNA stab04 B GuGAcGAGGAuGAuGAGGATT B 636 sense 455UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 457U21 siNA stab04 BGGucuGuuAuuGccAAGcATT B 637 sense 706 AUCUGCAGGAGUCUUCAAAAGCC 601 MYB:708U21 siNA stab04 B cuGcAGGAGucuucAAAAGTT B 638 sense 1051 AGGUGCUACCAACACAGAACCAC 602 MYB: 1053U21 siNA stab04 BGuGcuAccAAcAcAGAAccTT B 639 sense 146 CAGUGACGAGGAUGAUGAGGACU 599 MYB:166L21 siNA (148C) uccucAucAuccucGucAcTsT 640 stab05 antisense 455UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 475L21 siNA (457C)uGcuuGGcAAuAAcAGAccTsT 641 stab05 antisense 706 AUCUGCAGGAGUCUUCAAAAGCC601 MYB: 726L21 siNA (708C) cuuuuGAAGAcuccuGcAGTsT 642 stab05 antisense1051  AGGUGCUACCAACACAGAACCAC 602 MYB: 1071L21 siNA (1053C)GGuucuGuGuuGGuAGcAcTsT 643 stab05 antisense 146 CAGUGACGAGGAUGAUGAGGACU599 MYB: 148U21 siNA stab07 B GuGAcGAGGAuGAuGAGGATT B 644 sense 455UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 457U21 siNA stab07 BGGucuGuuAuuGccAAGcATT B 645 sense 706 AUCUGCAGGAGUCUUCAAAAGCC 601 MYB:708U21 siNA stab07 B cuGcAGGAGucuucAAAAGTT B 646 sense 1051 AGGUGCUACCAACACAGAACCAC 602 MYB: 1053U21 siNA stab07 BGuGcuAccAAcAcAGAAccTT B 647 stab07 sense 146 CAGUGACGAGGAUGAUGAGGACU 599MYB: 166L21 siNA (148C) uccucAucAuccucGucAcTsT 648 stab11 antisense 455UUGGUCUGUUAUUGCCAAGCACU 600 MYB: 475L21 siNA (457C)uGcuuGGcAAuAAcAGAccTsT 649 stab11 antisense 706 AUCUGCAGGAGUCUUCAAAAGCC601 MYB: 726L21 siNA (708C) cuuuuGAAGAcuccuGcAGTsT 650 stab11 antisense1051  AGGUGCUACCAACACAGAACCAC 602 MYB: 1071L21 siNA (1053C)GGuucuGuGuuGGuAGcAcTsT 651 stab11 antisense Myb Synthetic Modified siNAconstructs Target Seq ID Cmpd # Alias Sequence Seq IDAGGUGCUACCAACACAGAACCAC 602 31028 MYB: 1053U21 siNA senseGUGCUACCAACACAGAACCTT 633 AACAACCACAAAAUACAACAAGA 605 30977 MYB: 148U21siNA stab04 sense B GuGAcGAGGAuGAuGAGGATT B 636 AACAACCACAAAAUACAACAAGA605 30978 MYB: 457U21 siNA stab04 sense B GGucuGuuAuuGccAAGcATT B 637AACAACCACAAAAUACAACAAGA 605 30979 MYB: 708U21 siNA stab04 sense BcuGcAGGAGucuucAAAAGTT B 638 AACAACCACAAAAUACAACAAGA 605 30980 MYB:1053U21 siNA stab04 sense B GuGcuAccAAcAcAGAAccTT B 639AACAACCACAAAAUACAACAAGA 605 30981 MYB: 166L21 siNA (148C) stab05uccucAucAuccucGucAcTsT 640 antisense AACAACCACAAAAUACAACAAGA 605 30982MYB: 475L21 siNA (457C) stab05 uGcuuGGcAAuAAcAGAccTsT 641 antisenseAACAACCACAAAAUACAACAAGA 605 30983 MYB: 726L21 siNA (708C) stab05cuuuuGAAGAcuccuGcAGTsT 642 antisense AACAACCACAAAAUACAACAAGA 605 30984MYB: 1071L21 siNA (1053C) stab05 GGuucuGuGuuGGuAGcAcTsT 643 antisenseCAGUGACGAGGAUGAUGAGGACU 599 31025 MYB: 148U21 siNA senseGUGACGAGGAUGAUGAGGATT 630 UUGGUCUGUUAUUGCCAAGCACU 600 31026 MYB: 457U21siNA sense GGUCUGUUAUUGCCAAGCATT 631 AUCUGCAGGAGUCUUCAAAAGCC 601 31027MYB: 708U21 siNA sense CUGCAGGAGUCUUCAAAAGTT 632 CAGUGACGAGGAUGAUGAGGACU599 31101 MYB: 166L21 siNA (148C) UCCUCAUCAUCCUCGUCACTT 603 antisenseUUGGUCUGUUAUUGCCAAGCACU 600 31102 MYB: 475L21 siNA (457C)UGCUUGGCAAUAACAGACCTT 604 antisense AUCUGCAGGAGUCUUCAAAAGCC 601 31103MYB: 726L21 siNA (708C) CUUUUGAAGACUCCUGCAGTT 634 antisenseAGGUGCUACCAACACAGAACCAC 602 31104 MYB: 1071L21 siNA (1053C)GGUUCUGUGUUGGUAGCACTT 635 antisense Uppercase = ribonucleotide u, c= 2′-deoxy-2′-fluoro U, C T = thymidine B = inverted deoxy abasic s= phosphorothioate linkage A = deoxy Adenosine G = deoxy Guanosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically-modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′- S/AS ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually AS linkages“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoro Ribo — 1at 3′-end Usually AS “Stab 6” 2′-O- Ribo 5′ and 3′- — Usually S Methylends “Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′ and 3′-— Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O- 2′-O- 5′ and 3′-Usually S Methyl Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′-Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro*2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl*CAP = any terminal cap, see for example FIG. 10. All Stab 00-28chemistries can comprise 3′-terminal thymidine (TT) residues All Stab00-28 chemistries typically comprise about 21 nucleotides, but can varyas described herein. S = sense strand AS = antisense strand *Stab 23 hasa single ribonucleotide adjacent to 3′-CAP *Stab 24 and Stab 28 have asingle ribonucleotide at 5′-terminus *Stab 25, Stab 26, and Stab 27 havethree ribonucleotides at 5′-terminus p = phosphorothioate linkage

TABLE V Wait Time* Wait Time* Wait Time* Reagent Equivalents Amount DNA2′-O-methyl RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Wait Time* Equivalents: DNA/ Amount: DNA/2′-O- Wait Time*2′-O- Wait Time* Reagent 2′-O-methyl/Ribo methyl/Ribo DNA methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleicacid molecule comprises a sense strand and a separate antisense strand,each strand having one or more pyrimidine nucleotides and one or morepurine nucleotides; (b) each strand of the nucleic acid molecule isindependently 18 to 27 nucleotides in length; (c) an 18 to 27 nucleotidesequence of the antisense strand is complementary to a human Myc RNAsequence comprising SEQ ID NO:687; (d) the sense strand has a Stab 7stabilization chemistry configuration, wherein each pyrimidinenucleotide has a 2′-fluoro modification, each purine nucleotide has a2′-deoxy modification and both the 5′- and 3′-ends have a terminal capmoiety; (e) the antisense strand has a Stab 11 stabilization chemistryconfiguration, wherein each pyrimidine nucleotide has a 2′-fluoromodification, each purine nucleotide has a 2′-deoxy modification and theantisense strand has a phosphorothioate internucleotide linkage at the3′-end; and (f) wherein the nucleic acid molecule mediates RNAinterference. 2-14. (canceled)
 15. The nucleic acid molecule of claim 1,wherein the terminal cap moiety is an inverted deoxy abasic moiety. 16.The nucleic acid molecule of claim 1, wherein said nucleic acid moleculeincludes two or more phosphorothioate internucleotide linkages. 17.(canceled)
 18. The nucleic acid molecule of claim 1, wherein the 5′-endof the antisense strand includes a terminal phosphate group.
 19. Thenucleic acid molecule of claim 1, wherein the sense strand, theantisense strand, or both the sense strand and the antisense strandinclude a 3′-overhang.
 20. A composition comprising the nucleic acidmolecule of claim 1, in a pharmaceutically acceptable carrier ordiluent.
 21. The nucleic acid molecule of claim 19, wherein both thesense and the antisense strand include a 3′-overhang.
 22. The nucleicacid molecule of claim 19, wherein the 3′-overhang comprises 1-4nucleotides in length.
 23. The nucleic acid molecule of claim 19,wherein the 3′-overhang comprises thymidine residues.
 24. The nucleicacid molecule of claim 19, wherein the overhang comprisesribonucleotides.
 25. The nucleic acid molecule of claim 19 wherein theoverhang comprises ribonucleotides that are chemically modified at anucleic acid sugar, base, or backbone.
 26. The nucleic acid molecule ofclaim 1, comprising one or more blunt ends.